2653
Development 126, 2653-2662 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
DEV7690
Regulation of the spalt/spalt-related gene complex and its function during
sensory organ development in the Drosophila thorax
José F. de Celis1,*, Rosa Barrio2 and Fotis C. Kafatos2
1Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH,
2European Molecular Biology Laboratory, Meyerhofstrasse, 69117 Heidelberg, Germany
UK
*Author for correspondence (e-mail: [email protected])
Accepted 7 April; published on WWW 19 May 1999
SUMMARY
The nuclear proteins Spalt and Spalt-related belong to a
conserved
family
of
transcriptional
regulators
characterised by the presence of double zinc-finger
domains. In the wing, they are regulated by the secreted
protein Decapentaplegic and participate in the positioning
of the wing veins. Here, we identify regulatory regions in
the spalt/spalt-related gene complex that direct expression
in the wing disc. The regulatory sequences are organised in
independent modules, each of them responsible for
expression in particular domains of the wing imaginal disc.
In the thorax, spalt and spalt-related are expressed in a
restricted domain that includes most proneural clusters
of the developing sensory organs in the notum, and
are regulated by the signalling molecules Wingless,
Decapentaplegic and Hedgehog. We find that spalt/spaltrelated participate in the development of sensory organs in
the thorax, mainly in the positioning of specific proneural
clusters. Later, the expression of at least spalt is eliminated
from the sensory organ precursor cells and this is a
requisite for the differentiation of these cells. We postulate
that spalt and spalt-related belong to a category of
transcriptional regulators that subdivide the thorax into
expression domains (prepattern) required for the localised
activation of proneural genes.
INTRODUCTION
individually, are broader than the proneural clusters (GómezSkarmeta et al., 1996; Haenlin et al., 1997).
The overall growth of the wing disc is under the control of
several signalling molecules encoded by hedgehog (hh),
wingless (wg) and decapentaplegic (dpp) (Blair, 1995). These
genes also play critical roles during the patterning of sensory
organs (Phillips and Whittle, 1993; Mullor et al., 1997),
although their effects on achaete and scute expression might
be mediated by transcriptional regulators downstream of these
signalling molecules. In the patterning of veins, Hh activates
the expression of the homeobox genes of the iroquois gene
complex in the presumptive region of the L3 vein (GómezSkarmeta and Modolell, 1996) and Dpp activates the
expression of the structurally related genes spalt (sal) and
spalt-related (salr) in a central domain in the wing (de Celis
et al., 1996; Nellen et al., 1996; Lecuit et al., 1996). sal and
salr encode transcription factors with spaced sets of double
zinc-finger motifs (Kühnlein et al., 1994; Barrio et al., 1996)
and participate in the correct spacing of the wing veins (de
Celis et al., 1996). Elimination of both genes during
development of the wing results in phenotypes that are stronger
than those resulting from elimination of only sal, suggesting
that the genes have complementary roles. sal and salr are also
expressed in other regions of the wing disc that correspond to
the presumptive thorax, hinge and pleura, but their roles there
have not been yet established.
The Drosophila thorax is covered by two different types of
sensory organs, macrochaetae and microchaetae, distributed in
a characteristic pattern. They are formed by the progeny of
epidermal cells specified during larval (macrochaetae) and
pupal (microchaetae) development in the presumptive thorax
of the wing disc (Campuzano and Modolell, 1992). The
immediate sensory organ precursor (SOP) cells are singled out
amongst a cluster of potential precursor cells by the activity of
the proneural genes achaete and scute, two genes that encode
basic helix-loop-helix proteins (bHLH; Campuzano and
Modolell, 1992). Position-specific enhancers (Ruiz-Gómez
and Modolell, 1987; Gómez-Skarmeta et al., 1995) direct
expression of both achaete and scute in clusters of cells at
positions that prefigure the pattern of sensory organs. The
identification of genetic elements participating in the
regulation of achaete and scute is therefore a critical step in
understanding the generation of the pattern of sensory organs.
It is thought that achaete and scute expression is regulated in
individual proneural clusters by the combinatorial effect of
several genes that constitute a ‘prepattern’ of transcriptional
regulators (Stern, 1954; Modolell and Campuzano, 1998).
Several genes, such as the iroquois gene complex and pannier,
have been found to be members of this postulated prepattern.
They are expressed in the thorax in restricted domains which,
Key words: spalt, Gene regulation, Sensory organ development,
Drosophila
2654 J. F. de Celis, R. Barrio and F. C. Kafatos
In this work, we identify regulatory regions that direct
sal/salr expression at specific regions of the wing disc, and
show that hh, dpp and wg regulate these genes in the thorax.
Furthermore, we implicate sal and salr in the patterning of
sensory elements in the thorax. The expression of sal and salr
here occurs in a restricted domain that includes the position of
several macrochaetae proneural clusters. The analysis of
genetic mosaics of cells lacking both sal and salr indicates that
the function of these genes is required for the formation of a
subset of macrochaetae. We also show that expression of Sal
is specifically eliminated from the SOPs soon after they can be
identified, and that ectopic expression of sal or salr in these
SOPs prevents their differentiation.
MATERIALS AND METHODS
Drosophila strains
Flies were raised on standard Drosophila medium at 25°C. Mutations
not described in the text and balancer chromosomes can be found in
Lindsley and Zimm (1992). We used the reporter lines ara-lacZ
(irorF209; Gómez-Skarmeta et al., 1996), emc-lacZ (emcP5C; Garrell
and Modolell, 1990) and neu-lacZ (neuA101; Ghysen and O’Kane,
1989); the Gal4 lines Gal4-253, Gal4-MS209 and Gal4-765 (GómezSkarmeta and Modolell, 1996); and the UAS lines UAS-wg (Klein et
al., 1998), UAS- hh (Guillén et al., 1995), UAS-ci (Dominguez et al.,
1996), UAS-dpp (Staehling-Hampton and Hoffmann, 1994), UASlacZ (Brand and Perrimon, 1993), UAS-sal and UAS-salr (de Celis
et al., 1996). The sal mutants used were Df(2L)32FP-5, a small
deficiency that deletes both sal and salr (de Celis et al., 1996), and
FCK-25, a translocation with a breakpoint 5′ to the salr gene (data
not shown). The wingless mutations used were wgspd, wgCX3 and
wgCX4. We also used the following recombinant chromosomes:
tkv5w125 FRT40A (a gift from I. Rodriguez), pkaB3 FRT40A (Li et al.,
1995), pkaB3 dppd12 FRT40A (Pan and Rubin, 1995) and armLacZ
FRT40A.
Identification of sal/salr regulatory regions in the wing
disc
Individual EcoRI fragments originated from digestion of the phages
located between the breakpoints of FCK-25 and FCK-68 mutations
(except the ones covering the coding region – phage G3.1 described
in Frei et al., 1988, Fig. 2) were subcloned into the same restriction
site of the enhancer tester C4PLZ vector (Wharton and Crews, 1993).
These fragments were placed in front of a weak P-element promoter
attached to the nuclear lacZ gene. The vector also contains the miniwhite gene as an eye colour marker. The constructs generated were
introduced in the germline by P-element transformation as described
(Rubin and Spradling, 1982). The β-gal expression was detected by
immunostaining of 3rd instar larval tissues. For each construct, at least
three independent lines were tested.
Generation of mosaics
Gal4-expressing clones
The expression of Gal4 was induced in individual cells using a
combined Gal4-Flip out system (de Celis and Bray, 1997). Clones
were labelled by the presence of β-gal, and the expression of sal was
monitored in clones of cells that express the proteins Wg, Hh, Dpp
and Ci using the corresponding UAS lines. The genotypes of the
larvae were f36a hsFLP1.22; P[Ubx/abxFRT f+ FRTGal4-β-gal]/UASwg (or UAS-hh, UAS-dpp, UAS-ci). Clones were induced by 7 minutes
heat shock at 37°C; 48-72 hours after egg laying.
Df(2L)32FP5 clones
Mutant cells homozygous for a deficiency of sal and salr
(Df(2L)32FP5) were induced by X-ray-mediated mitotic
recombination in flies of genotype f36a; Df(2L)32FP5/ M(2)30
In(f+)35B. Clones were labeled by the homozygosity of the cell
marker forked.
FLP/FRT clones
Mitotic recombination was induced by 1 hour heat shock at 37°C
in larvae of the following genotypes: hsFLP1.22; tkv5w125
FRT40A/armLacZ FRT40A, hsFLP1.22; pkaB3 FRT40A/ armLacZ
FRT40A and hsFLP1.22; pkaB3 dppd12 FRT40A/ armLacZ FRT40A.
Mutant clones were visualised in third instar larvae by the absence of
β-gal expression.
Generation of antisera
Two fragments of spalt cDNA (from amino acid 403 to 515 and from
803 to 936; Kühnlein et al., 1994) subcloned in frame into the pRSET
C vector (Invitrogen; these subclones were kindly provided by R.
Shuh) were expressed in BL21 cells. The resulting truncated Sal
proteins were purified according to the manufacturer instructions and
injected together in rats and rabbits following standard protocols.
Immunocytochemistry
Imaginal discs were stained with anti-β-gal antibodies following
standard procedures. Rabbit anti-β-gal from Cappell was used at a
final 1/5000 dilution in PBT-BSA (PBS, 0.5% Triton X-100, 1%
BSA). For double staining, rabbit anti-β-gal (1/1000) and rat anti-Sal
(1/200) antibodies were used. We also used mouse anti-Ac (1/20),
mouse anti-En (1/20), mouse anti-β-gal (1/500), mouse anti-Wg
(1/5000), rabbit anti-Sal (1/200) and rat anti-Ci (1/100) antibodies.
Secondary antibodies were from Jackson Inmunological Laboratories
and were used at a final 1/200 dilution. Samples were analysed with
a Zeiss Axiophot for HRP staining or a Leika TCS confocal
microscope for double staining with secondary fluorescence
antibodies.
RESULTS
Molecular analysis of sal and salr regulation in the
wing disc
The genes sal and salr are expressed in identical patterns in the
wing disc (de Celis et al., 1996). The territory of expression
includes a central stripe in the wing blade, anterior and
posterior pleural regions and a subset of the proximal hinge
and thorax (Fig. 1B). The region necessary to drive both sal
and salr expression in the thorax and some domains of the wing
is approximately 60 kb in size, and is located between the
breakpoints of two translocations, FCK-25 and FCK-68 (Fig.
1 and data not shown). We cloned this DNA in fragments of
0.3 to 10 kb in front of the reporter lacZ gene to uncover the
relevant regulatory elements in the wing disc. Several
fragments direct expression of β-gal in places where sal and
salr are present in the thorax (AS, ABO, ABI and LA), hinge
(EME, AL, LA), pleura (BE, EME, AK, BI) and wing blade
(BE, EME, BO, LA, BI, AK; Fig. 1). In most cases, the
expression of β-gal occurs both in subdomains where sal and
salr are normally expressed and in specific ectopic domains
(Fig. 1). For each construct, β-gal expression patterns were
identical in (at least) three independent transformant lines,
demonstrating that the complexity of each pattern is generated
by the driving DNA, and is not due to insertion position effects
(data not shown). For instance, the constructs ABO and ABI,
which overlap by approximately 2.2 kb, reproduce the
sal and salr in Drosophila macrochaetae pattern formation 2655
Fig. 1. Regulatory regions of sal and salr in the
wing disc. The upper part of the figure is a
simplified molecular map of the sal/salr gene
complex indicating the location of coding regions
(horizontal arrows). It shows in scale the position
and relative sizes of the different DNA fragments
that we have use to drive β-gal expression in the
wing disc (colored boxes). The genes are drawn
with grey boxes representing exons, V-shaped lines
symbolising introns and open boxes indicating 5′
and 3′ UTRs. Short vertical lines indicate EcoRI
restriction sites in the genomic DNA. The vertical
arrows indicate the positions of breakpoints for
two mutations (FCK-25 and FCK-68), which are
approximately 60 kb apart and delimit the
regulatory regions required for normal salr
expression in the wing and thorax and sal
expression in the thorax (data not shown). Below,
the black and white pictures show expression of βgal in discrete regions of the wing disc in flies
transformed with the identified constructs,
compared with the endogenous expression pattern
of sal (Sal). Note that some overlapping constructs,
such as BI and AK, drive β-gal to the same
regions. However, the expression driven by LA in
the thorax and hinge must be directed by regions
of the construct not overlapping with the BO
fragment, while the expression in the wing pouch
(region between the longitudinal vein L2 and the
wing margin) must be directed by the genomic
DNA present in both constructs. (A-D) Schematic
representations of wing imaginal discs. (A) The
regions in the disc that will give rise to the adult
wing (w), hinge (h), pleura (p) and thorax (t,
yellow), which has been divided into four areas by
dashed lines: the posterior En-expressing region
(1; Fig. 2A), the anterior stripe coincident with
maximal accumulation of Ci (2; Fig. 2B), the lateral thorax (3) and the central thorax (4). The blue line represents the anterior/posterior
compartment boundary (A/P) and the red line the dorsal/ventral one (D/V). (B) The pattern of endogenous expression of sal and salr is shown
in green. (C) Some examples of transgenic expression of β-gal from the different constructs following the color code as in the upper scheme.
Some of the β-gal domains overlap in certain regions (stripes). (D) Superimposition of the Sal expression pattern and the summed expression of
β-gal in all the lines; it shows that the combined β-gal expression from all the tested constructs covers almost the entire Sal domain, but also
extends ectopically. Co-expression is shown in yellow-orange, expression of β-gal outside the Sal expression domain in red, and expression of
Sal where no β-gal was found in green.
endogenous expression of sal/salr in the posterior
compartment of the thorax (Fig. 1); they also express β-gal in
the anterior pleura (Fig. 1), a place where these genes are not
normally transcribed. Similarly, the construct LA is expressed
in a pattern that includes part of the domain of endogenous
sal/salr expression in the thorax and also in an anterior ectopic
domain in the wing blade (Fig. 1). Several constructs located
5′ to the salr gene or in the large intron upstream of its coding
region (BE, EME) direct generalised expression of β-gal in the
wing blade, in a pattern that includes the stripe of normal
sal/salr expression but also adjacent ectopic anterior and
posterior regions (Fig. 1). The observation that consistent
expression of β-gal is driven by several constructs in places
where sal and salr are not expressed suggests that the
regulation of these genes involves the interplay of both
activating and repressing regulatory sequences. The combined
analysis of these β-gal constructs, in a total of 92 independent
transgenic lines, allows a broad localisation of multiple DNA
sequences responsible for sal and salr expression in the wing
disc (Fig. 1). It also suggests that the expression of sal/salr in
the different parts of the wing disc is regulated in an
independent manner. We have shown previously that, in the
wing blade, these two genes are regulated by Dpp (de Celis et
al., 1996), and the present molecular analysis predicts that the
expression in thorax, hinge or pleura is achieved by a different
set of factors (see below).
Wg, Dpp and Hh are implicated in the regulation of
sal/salr expression in the wing thoracic region
The sal and salr genes are expressed in only part of the thorax,
in three domains (1, 2 and 3 in Fig. 1A), which were defined
with reference to en, wg and ci: the thoracic posterior
compartment marked by En (Figs 1A, 2A), an adjacent stripe
anterior to the anteroposterior compartment boundary
corresponding to the stripe of maximal accumulation of Ci
(Figs 1A, 2B), and a zone between the stripe of wg expression
and the hinge (Figs 1A, 2C). A fourth domain (4 in Fig. 1A),
the central thorax from where only microchaetae develop, does
2656 J. F. de Celis, R. Barrio and F. C. Kafatos
Fig. 2. Effects on Sal expression of ectopic expression of Hh, Ci and
Dpp in the thorax. (A-C) Expression of Sal (green) in the thorax in
relation to En (red, A), Ci (red, B) and Wg (red, C). (D) Expression
of Sal in late third instar. (E) Ectopic expression of Sal (green) in
clones of cells expressing hh (<hh>, red). Arrowhead points to a hhexpressing clone in the central thorax that is accompanied by ectopic
Sal expression, both in the clone itself (yellow) and in the
surrounding cells (green). (F,G) Induction of Sal expression in the
wing blade (F, yellow, arrowhead), but not in the hinge (F, red,
arrow) or central thorax (G, red, arrow) in Dpp-expressing clones
(<dpp>, labelled in red). (H,I) Effects on Sal expression of ectopic
expression of Ci (<Ci>, red). Ci only induces Sal expression in the
wing blade (H, arrowhead), but not in the hinge (H, arrow) or central
thorax (H, lower arrow and I, arrow). Sal expression in Ci-expressing
cells is manifest as yellow-orange, and its absence as red.
Fig. 3. Effects on Sal expression of
elimination of Pka, Pka and Dpp, and
reduction of Tkv. Sal expression (green; A-D
and A″-D″) and β-gal expression (red; A-D
and A′-D′). Mutant clones are evident by the
absence of red staining. (A-A″) Ectopic
expression of Sal in a Pka clone localised in
the central thorax (arrow). Sal expression
remains unchanged (arrowhead) in a mutant
clone generated in thoracic region 2 (see Fig.
1). (B-B″) Expression of Sal in the thorax in
Pka, dpp double mutant clones. Due to the
absence of dpp, Pka mutant clones are now
incapable of inducing sal ectopic expression
in the central thorax (arrowhead). In other
thoracic regions, the double mutant cells show
a reduction in the level of expression of
Sal/Salr (arrow). (C-C″) Example of tkv5w124
clone that causes a reduction in Sal expression
in region 2 (arrow), but do not modify this
expression in other areas (arrowhead). (D-D″)
The reduction of Tkv levels does not modify
the sal expression levels in the central thorax
or in region 3 (arrowheads).
not express sal/salr. To explore the regulatory mechanisms that
localise sal/salr expression with respect to the anteroposterior
compartment boundary and wg, we first performed
experiments in which genes that function in developmental
signalling were expressed ectopically using the Gal4 system
(Brand and Perrimon, 1993; see Materials and Methods).
A series of experiments led to the conclusion that, in the thorax,
dual hh signalling is required to induce sal/salr expression:
signalling through dpp and signalling that is dpp independent.
Thus, expression of hh in clones within the central thorax
(presumably accompanied by induction of dpp) leads to ectopic
expression of sal/salr; interestingly, this ectopic expression is
observed both in hh-expressing cells and in adjacent cells (Fig.
2E). In contrast, ectopic expression of dpp does not result in
activation of sal transcription in the thorax (Fig. 2G) or in the
hinge (Fig. 2F), but it does so in the wing blade (Fig. 2F). We
also mis-expressed in clones of cells the transcription factor
Cubitus interruptus (Ci), a key mediator of Hedgehog signalling
(Ruiz i Altaba, 1997). We find that Ci is only able to activate sal
ectopically in the wing blade (Fig. 2H), a place where ectopic
expression of Ci results in novel expression of dpp (Dominguez
et al., 1996), but not in the central thorax or wing hinge (Fig. 2H,
I). In any other tissue studied to date, Hh signalling depends on
Ci; since hh positively regulates sal in the thorax, the failure of
ectopic Ci to activate sal expression there may be ascribed to the
presence of countermanding repressors (see below).
However, even though dpp is not sufficient to induce sal/salr
in the thorax, it is required. Thus, mitotic clones of Pka
(corresponding to constitutive activation of hh signalling; Li et
al., 1995; Pan and Rubin, 1995) show cell autonomous
expression of sal (Fig. 3A); in contrast, Pka, dpp double mutant
clones do not express sal (Fig. 3B), indicating that, close to the
Dpp source, hh and dpp signalling must cooperate to activate
sal expression in the thorax. In agreement with this, we also
find that the expression of sal can be reduced in tkv mutant
cells, which have reduced level of a Dpp receptor (Fig. 3C,D).
sal and salr in Drosophila macrochaetae pattern formation 2657
The requirement of dpp function for induction of sal differs in
different parts of the thorax. In the central thorax, where sal is
not expressed normally, tkv clones have no effect (Fig. 3D,
arrowhead). In region 2 (Fig. 1A), where dpp is expressed
normally, tkv clones result in reduced expression of sal (Fig.
3C, arrow). In other regions of the thorax, expression of sal is
unaffected by the reduction of tkv (Fig. 3C,D, arrowheads).
A prominent stripe of wg expression is seen in the thorax,
within the sal non-expressing region 4 and close to the border
of the sal-expressing region 3 (Figs 1A, 4A,C). This
observation raised the possibility that wg may act as a repressor
of sal expression in the thorax, and possibly in other regions
of the wing disc. Indeed, in thoracic region 3, sal expression
is repressed in and around clones of cells that overexpress wg
(Fig. 4F,G). Furthermore, wg overexpression in the hinge
region (in Gal4-MS209/UAS-wg) results in a reduction of sal
expression (Fig. 4E), whereas a reduction of wg expression in
the hinge of imaginal discs as a result of the regulatory
mutation spade flag (wgspd) results in a consistent increase in
sal expression (Fig. 4B). Reduction of wg expression in the
thorax in the heteroallelic combination wgCX3/wgCX4 results in
the expansion of Sal expression (Fig. 4D). However, Sal is not
expressed in all region 4, indicating that a repressor other than
wg is responsible for the exclusion of Sal in this region.
It is likely that both sal activation by hh/dpp and its
repression by wg are mediated by the regulatory regions that
we identified as responsible for direct sal/salr expression in the
thorax (Fig. 1). As described before, the expression of β-gal
driven by the sal/salr regulatory regions occurs both in the
normal domain of sal/salr expression and also in some ectopic
territories (Figs 1, 5A,C). The expression of β-gal in the
transgenic line LA is included in the territory of sal/salr
expression in the thorax (Fig. 5D). Much of this expression is
removed when wg is expressed ectopically (Fig. 5E,F). We
used two Gal4 drivers, Gal4-253, which is expressed in all
proneural clusters and in the SOPs (not shown), and Gal4-756,
which is expressed homogeneously at a lower level throughout
the thorax (Gómez-Skarmeta and Modolell, 1996; data not
shown). When wg overexpression is generalised and at low
level (Gal4-756/UAS-wg), the expression of the LA enhancer
is limited to the dorsal part of thoracic region 2 (Fig. 5E). When
wg overexpression is stronger but focused in the region of
macrochaetae precursors (Gal4-253/UAS-wg; see below), the
expression of the LA enhancer is only seen in a ventral part of
region 2 (Fig. 5F). These results confirm the repressive action
of wg and indicate that it is mediated by sequences contained
within the LA fragment. However, in the same experiments,
the endogenous sal expression is unaffected by wg
overexpression (Fig. 5E,F), suggesting that the levels or time
of expression of ectopic wg are ineffective in repressing
sal/salr expression. Therefore, we suggest that, in these
experiments, the inhibitory action of wg can be counteracted
by sequences of the endogenous regulatory region located
outside the LA fragment. Consistent with a requirement for
both hh and dpp signalling to activate sal/salr in the thorax, the
expression of β-gal in the line LA is not modified when dpp is
expressed ectopically (data not shown).
Relationships between sal expression and sensory
organ patterning in the thorax
The domain of sal/salr expression in the thorax was mapped
with more precision with respect to the emerging Sensory
Organ Precursor cells (SOP), which can be identified in the
disc using the reporter line neuralised-lacZ (neu-lacZ; Ghysen
and O’Kane, 1989). The sensory organs included in the sal/salr
domain are most of the lateral macrochaetae (ANP, PNP, ASA,
PSA, APA, PPA) and also the ASC, PSC and PDC
macrochaetae (Fig. 6E,F; see figure legend for abbreviations);
the PS and ADC macrochaetae arise outside this domain. As
shown above, sal and salr are not expressed in the central
domain of the thorax, the region from which most of the
microchaetae will develop during pupal development. This was
confirmed by demonstrating that extramacrochaetae (emc), a
negative regulator of the ac and sc genes that marks the
microchaetae territory, is expressed in a domain nearly
Fig. 4. Effects of wg on Sal expression in the thorax. In all panels,
Wg is shown in red and Sal in green. (A) Expression of Wg and Sal
in wild-type late third instar wing disc. The limit of Sal expression is
slightly more lateral than the Wg expression region in the central
thorax. (B) Expression of Wg and Sal in wgspd mutant wing disc. In
this genotype, wg expression is strongly reduced in the hinge, where
sal expression is now enhanced (arrow). (C) Expression of Wg and
Sal in a wild-type thorax. (D) Expression of Wg is almost absent in
the heteroallelic combination wgCX3/wgCX4 and Sal expression
extends into more lateral regions. (E) Expression of Wg and Sal in
Gal4-MS209/UAS-wg wing disc, showing a strong reduction of sal
expression in the expanded hinge region (arrow). (F) Sal expression
is reduced at and around clones of cells that express wg ectopically
(labelled in red, see Material and Methods). This repression is clearly
evident in the lateral thorax (arrowheads), whereas within the Sal
expression domain in the wing pouch ectopic wg does not repress sal
(arrow; yellow). (G) Higher magnification of the thoracic region of a
disc carrying wg-expressing clones. Below, single green channel
showing the absence of Sal in the clones, and its reduction in
immediately surrounding cells.
2658 J. F. de Celis, R. Barrio and F. C. Kafatos
complementary to sal/salr (Fig. 6C). Sal is expressed in the
promote SOP development. In addition, stronger ectopic
domain that encompasses most of the macrochaetae proneural
expression of salr using the UAS line UAS-salr2 caused the
cell clusters (Fig. 6A,B). Interestingly, when specific cells of
absence or size reduction of some macrochaetae (Fig. 7F).
the proneural clusters are ‘singled out’ to form the SOP, sal
These contradictory effects on the pattern of macrochaetae
expression is eliminated from these cells and their descendants
when sal/salr are ectopically expressed were associated with
(Fig. 6G,H). This is a requisite for SOP development, because
defective localisation of proneural clusters in the imaginal disc.
when sal expression is experimentally maintained in SOP cells
Thus, generalised low expression of salr (UAS-salr1) causes a
they do not differentiate (see below).
weak expansion of the APA proneural cluster (Fig. 7I), whereas
The localisation of most macrochaetae SOPs within the
higher levels of generalised salr expression (UAS-salr2) result
territory of sal/salr expression in the thorax, and the dynamics
in a broader rearrangement of proneural clusters, including a
of sal expression associated with proneural clusters (Sal+) and
larger expansion of the DC and NP (which now appear fused),
SOPs (Sal−), raise the possibility of a function for the sal/salr
and the apparent loss of the PA proneural cluster (Fig. 7J).
genes in sensory organ patterning. The requirement of sal and
These results indicate that the restriction and levels of sal/salr
salr was studied by inducing clones of cells homozygous for a
expression participate in the positioning of proneural clusters
deficiency, Df(2L)32FP5, that includes both genes. Mutant
in the thorax.
clones in the thorax, marked with forked, were viable and of
Combinations between Gal4-253 (which strongly targets
normal size, indicating that sal and salr are not required for the
expression to all proneural clusters and SOPs) with UAS-sal,
viability of thoracic cells (data not shown). Loss of sal/salr
UAS-salr1 or UAS-salr2 have dramatic effects on the
caused the absence of two macrochaetae, the ANP and PNP,
macrochaetae (Fig. 7G and data not shown). Only APA formed
and also the anterior displacement of the PSC and PDC, which
normally; the others either disappeared or differentiated as
differentiated abnormally close to their anterior counterparts;
extremely small bristles (ADC, PDC, PPA and PSC; Fig. 7G).
the remaining seven macrochaetae were unaffected (Fig. 7B).
The effects of ectopic sal or salr overexpression specifically in
A similar, albeit weaker, phenotype was observed in some
the macrochaetae SOPs were further investigated by following
allelic combinations in which sal and salr expression are
directly the formation of each SOP and its progeny in the wing
reduced. Thus, combinations involving any salr allele over the
disc. We found that, in Gal4-253/UAS-sal wing discs, all
deficiency of the complex caused the absence of the ANP
proneural clusters, where Sal is overexpressed, appear in their
macrochaetae (data not shown).
normal positions, and that later the SOPs are singled out normally
The FCK-25/Df(2L)32FP5 heteroallelic combination
results in the reduction of sal/salr expression. In this genetic
background, the ANP proneural cluster does not show ac
expression (Fig. 6D) and the ANP macrochaetae is not
formed (data not shown); these observations suggest that
sal/salr contribute to the normal expression of ac, which is
necessary for macrochaetae formation. However, most
proneural clusters develop normally despite reduction of
sal/salr function and many macrochaetae included in the
expression domain of sal/salr differentiate normally in the
total absence of these genes. Evidently, additional factors,
besides sal and salr, participate in the transcriptional
activation of ac/sc. These factors most likely correspond to
previously characterised regulators of ac/sc, such as the
genes of the iroquois complex (iro; Gómez-Skarmeta et al.,
1996), which have similar, albeit weaker, and partially
complementary effects to sal/salr on macrochaetae formation
when they are expressed ectopically (Fig. 7D,H and GómezSkarmeta et al., 1996). As expected from the expression
pattern of sal and salr, the differentiation of the microchaetae
was not affected either in heteroallelic combinations or in
homozygous Df(2L)32FP5 clones (Fig. 7B).
The influence of sal and salr on macrochaetae pattern
formation was also studied in experiments in which either Fig. 5. Comparison of Sal and β-gal protein localisation in several
of these genes was ectopically expressed in the thorax using reporter lines and effects of wg on LA reporter expression. Expression of
the Gal4 system. In the case of salr, we also used two endogenous Sal (green) and transgenic β-gal (red) in the reporter lines BE
different UAS lines, named UAS-salr1 and UAS-salr2, (A), AK (B), ABI (C) and LA (D-F). Expression of β-gal in each line
which produce low and high levels of Salr expression, occurs in places where Sal and Salr are normally expressed (thorax in the
lines LA and ABI; pleura in AK; wing blade in BE) and, in some cases,
respectively, in combination with any Gal4 driver (not also in positions where Sal/Salr are not expressed (anterior and posterior
shown). When sal or salr were expressed at low levels in domains in the wing blade in BE and vein L5 in AK). (E,E′,F) From LA
all thoracic cells (combinations between Gal4-756 with discs where wg is ectopically expressed (Gal4 lines 756 and 253,
UAS-sal or UAS-salr1) several extra macrochaetae respectively) resulting in reduced β-gal expression. E′ and inset in F
differentiated in ectopic positions (Fig. 7E,F and data not present the single red channels, which shows β-gal expression restricted to
shown), indicating that sal and salr have the capability to the most dorsal and lateral domains, respectively, of LA β-gal pattern.
sal and salr in Drosophila macrochaetae pattern formation 2659
Fig. 6. Expression of Sal in relation to sensory organs in the thorax
and effects on ac expression of changes in sal/salr activity. In the
upper part, a scheme (after Campuzano and Modolell, 1992) shows
the location of sensory organs in a third instar wing imaginal disc
(right). The region marked in blue will give rise to the adult thorax,
where the microchaetae are depicted as small circles and the
macrochaetae as labelled rectangles (left). The names of the
macrochaetae are: ADC and PDC, anterior and posterior
dorsocentral; ASC and PSC, anterior and posterior scutellars; ASA
and PSA, anterior and posterior supralar; ANP and PNP, anterior and
posterior notopleural; APA and PPA, anterior and posterior postalar;
PS, presutural. Other sensory organs in that region are tr1 and tr2
(sensillum tricoideum 1 and 2). Unlabelled black dots represent
sensory organs outside the thorax. (A,A′) Expression of Sal (green)
and Ac (red) in the thorax. Several macrochaetae proneural clusters
in the spalt expression domain are indicated by numbers: DC (1),
APA (2), PSA (3), ANP (4), PNP (5), anterior notal wing process (6),
Tegula (7) and SC (8). Note that the DC cluster spans the border
between the Sal-expressing region 2 and the non-expressing region 4.
(A′) A different focal plane of the same disc to show the scutellar
region. (B,B′) Single red channel showing Ac expression.
(C) Expression of Sal (green) and emc (red) in the emc reporter line
emcP5C. Sal expression is excluded from the central region of the
thorax, where emc is maximally accumulated (arrow).
(D) Expression of Ac in FCK-25/Df(2L)32FP5 discs is reduced in
the ANP cluster (arrow), the macrochaetae affected by reduction of
salr in this heteroallelic combination. (E,F) The wild-type domain of
Sal expression includes most macrochaetae sensory organ precursor
cells, but ADC is in the non-expressing central thorax and PDC is at
the border of the expression domain. Sal expression is shown in
green and β-gal expression in the SOP reporter line neu-lacZ is
shown in red. F is a different focal plane to show the scutellar region;
arrow points to PSC that has already divided. SOPs outside the
thoracic region are not indicated. (G) Expression of Sal (green) is
eliminated from emerging SOPs (red dots) and their progeny.
(H) Single green channel confirming the absence of Sal expression
(arrowheads). The scutellar region is shown at a different focal plane.
category of genes. sal and salr encode proteins characterised
by paired zinc-finger domains that have been conserved in
structure and sequence during evolution. So far, only the DNAbinding affinity of Drosophila Salr has been tested; the central
fingers of Salr show affinity for double-stranded A/T-rich
sequences of DNA (Barrio et al., 1996). The structural and
sequence conservation suggests that proteins of the Sal family
recognise similar DNA sequences, although no downstream
gene for any Sal protein has yet been identified.
but either do not divide or divide later than normal (Fig. 7K).
These observations indicate that the elimination of sal expression
from the SOPs observed in normal development is functionally
significant, being a requisite for later SOP differentiation.
DISCUSSION
The Drosophila thorax appears to be divided into domains of
expression of prepattern genes (Modolell and Campuzano,
1998; Calleja et al., 1996), which might be related with the
patterning of sensory organs. In this work, we have identified
the gene complex spalt/spalt-related as a member of this
Regulation of sal/salr expression
The expression of sal and salr in the thorax is restricted to a
broad domain that includes the positions where most proneural
clusters appear. As in many regulatory Drosophila genes, the
genomic regions directing sal and salr expression are organised
in modules, and we have been able to identify within a 60 kb
region distinct regulatory domains responsible for distinct
aspects of sal/salr expression in the thorax and other parts of
the wing disc. A surprising observation is that many of the
constructs consistently drive reporter gene expression in some
places where sal/salr are not normally expressed. Most likely
the genomic DNA used in such constructs contains enhancer
sequences able to activate sal/salr expression and lacks other
sequences that normally restrict spatially this activation. The
observed ‘ectopic’ expression is frequently combined with
2660 J. F. de Celis, R. Barrio and F. C. Kafatos
Fig. 7. Bristle phenotypes and Ac expression
associated with changes in Sal/Salr activity.
(A) Representation of a wild-type adult thorax
showing the location of the microchaetae (circles) and
macrochaetae (labelled rectangles). The names of the
macrochaetae are as in Fig. 6. In the remaining panels
(B-H), which summarise the effects of loss and
overexpression of macrochaetae regulatory factors,
blue rectangles represent macrochaetae that
differentiate in abnormal positions, red rectangles
represent eliminated macrochaetae, green rectangles
are ectopic macrochaetae and yellow rectangles
indicate macrochaetae showing abnormally small size.
Named macrochaetae are unaffected. (B) Thorax
summarising the phenotypes observed in 15 large and
overlapping Minute+ clones of a deficiency for sal and
salr, Df(2L)32FP5. Note that, in mutant clones, the
ANP and PNP macrochaetae are absent, and PSC and
PDC differentiate closer to their anterior counterparts.
(C) Ectopic expression of wg throughout the thorax
causes the elimination and mislocation of the indicated
chaetae. (D,H) Ectopic expression of araucan (a
member of the iroquois complex) either throughout
the thorax (using Gal4-756; (D) or targeted to the
proneural clusters and SOPs (Gal4-253; (H) causes
both elimination and ectopic formation of
macrochaetae (green rectangles). (E,F) Representation
of the positions where ectopic macrochaetae
differentiate when salr is expressed at low (E) or
higher (F) levels in the thorax. UAS-salr1 produces
lower levels of Salr ectopic protein than the UAS-salr2
line. (G) Elimination or abnormally small size
macrochaetae when Sal or Salr are expressed in
proneural clusters and SOPs in Gal4-253/UAS-sal and
Gal4-253/UAS-salr flies. Many microchaetae are also
eliminated in these two genotypes. (I,J) Expression of
Ac (green) when Salr is ectopically expressed at low
(I) or higher (J) levels. Note the expansion of the APA
cluster in I, and its loss in J (arrow) associated with a more extensive reorganization of proneural clusters. (K) Expression of Ac (green) and
sensory organ precursor cells (red) in a wing disc where Sal expression is high in proneural clusters and SOPs (Gal4-253/UAS-sal). The
scutellar region is shown at different focal plane. Note that SOP singling out and Ac expression of PSC are normal, but that PSC has not
divided (compare to the pair of progeny cells at the same stage in Fig. 6F).
expression in regions where the endogenous sal/salr genes are
expressed. Thus, we envision the regulation of sal and salr as
a complex process in which both transcriptional activators and
repressors interact with particular regulatory regions to generate
the normal restricted expression domains.
We do not know what genes participate directly in the
regulation of sal and salr, although we expect that future
refined characterisation of the putative sal/salr enhancers will
help to identify conserved binding sites and consequently
suggest candidate transcription factors implicated in sal/salr
Fig. 8. Model of sal/salr regulation in the thorax. The thoracic
expression of sal/salr (light green) is activated in the anterior
compartment by Hh (blue arrows) and Dpp (yellow arrows), and is
repressed by Wg (red bracket). We postulate that the combined
effects of hh and dpp signalling can overrule the repression mediated
by wg in cells closer to the anterior-posterior compartment boundary
(blue line). We also postulate that an additional repressor in the
central thorax (R) collaborates with wg in the repression of sal/salr.
This repression mechanism can only be alleviated upon ectopic hh
and dpp signalling, for example in Pka mutant clones (see Fig. 3A).
regulation. At this stage, it is clear that several signalling
molecules play critical roles in defining the territories of
sal/salr expression. Previous analysis showed that the stripe of
sal/salr expression in the wing is under direct regulation by the
TGF-β molecule Dpp (de Celis et al., 1996; Nellen et al., 1996;
Dpp
Wg
R
Hh
sal and salr in Drosophila macrochaetae pattern formation 2661
Lecuit et al., 1996). We have been able to identify fragments
of DNA directing expression of the reporter gene in the wing
blade, but none of them reproduces the restricted pattern of
endogenous expression of sal/salr. A 6.7 kb long DNA
fragment 5′ to the sal gene reproduces the expression of sal in
the wing blade, but smaller subfragments of this region do not
(Kühnlein et al., 1997). The fragments that we have analysed
in the equivalent region and in the first intron of salr are of
considerably smaller size, and consistently result in expression
of the reporter gene in ectopic positions of the wing blade.
Thus, it seems that a large 5′ regulatory region is responsible
for the generation of the restricted central domain of expression
of both sal and salr in the wing blade, and that both regions
include a combination of Dpp-responsive positive elements as
well as sites that mediate repression. Our analysis also shows
that most enhancers responsible for sal and salr expression in
other regions of the wing disc are located 3′ to each gene. The
level of resolution of the present analysis does not allow us to
address whether there are common enhancers for both sal and
salr or whether, alternatively, they are duplicated.
Some of the localised enhancers are regulated by factors
other than Dpp, and we identify Wingless and Hedgehog as two
additional signalling molecules that appear to participate in
regulating sal/salr in the thorax and wing hinge. sal is expressed
throughout the posterior thorax and in an adjacent sickle-shaped
anterior region that excludes the central thorax (Fig. 8). The
activation of sal by hh in the thorax requires the presence of
dpp, because sal is only ectopically expressed in Pka mutant
cells, but not in Pka, dpp double mutant cells. However, ectopic
expression of dpp is not enough to activate sal/salr outside of
its normal expression domain. We suggest that the requirement
for both a short-range signal (Hh) and a long-range signal (Dpp)
to activate sal/salr in the thorax contributes to the observed
restriction of sal/salr expression within the thorax. A
collaboration of hh and dpp signalling has also been observed
in the activation of the iro gene complex expression in the wing
blade (Gómez-Skarmeta and Modolell, 1996). We have shown
that wg overexpression in thoracic region 3 can repress sal
expression within a short range. Thus, the restricted pattern of
thoracic sal/salr expression may depend not only on positive
regulation by hh/dpp, but also on repression mediated by wg
signalling. It is notable that Wg is expressed in a stripe of cells
parallel to the central border of sal/salr expression (Fig. 8).
However, the position of this stripe of wg-expressing cells with
respect to the sal/salr expression domain is asymmetrical.
Furthermore, strong reductions in wg function do not result in
a complete expansion of sal expression in region 4. Therefore,
we suggest that additional factors present here help repress
sal/salr expression independently of wg (Fig. 8).
Both Hh and Wg signalling have dramatic effects on chaetae
pattern formation (Phillips and Whittle, 1993; Mullor et al.,
1997) and it is possible that some of these effects are mediated
by sal and salr. Interestingly, the expression of a vertebrate sal
homologue appears to be regulated by a hedgehog homologue
(Köster et al., 1997), raising the possibility that, not only the
structure, but also some aspects of the regulation of this family
of transcription factors has been conserved. In conclusion, the
regulatory sequences of sal/salr in the wing appear to integrate
the action of multiple signalling pathways and transcription
factors to generate the restricted expression of these genes. A
similar situation occurs during embryonic development, where
expression of sal is regulated by several maternal and gaps
genes (Kühnlein et al., 1997).
A role for sal/salr in macrochaetae patterning and
differentiation
The positioning and differentiation of sensory organs in the
thorax is a multistep process that is initiated by the activation
of proneural genes in clusters of cells named proneural
clusters. Within each proneural cluster, a characteristic number
of SOP cells are then singled out and undergo two differential
divisions each to generate the four cells that form each sensory
organ (Campuzano and Modolell, 1992). The normal
expression pattern of sal/salr and the results of manipulating
their activity indicate that these genes play significant roles in
both the generation of particular proneural clusters and the
differentiation of the SOPs. First, sal/salr expression is present
in the domain from which most proneural clusters will form.
Second, elimination of sal/salr activity in genetic mosaics
shows that the function of these genes is required for the
development of two notal macrochaetae (ANP, PNP) as well
as the fine positioning of two other macrochaetae (PDC, PSC).
Furthermore, some weak sal/salr allelic combinations cause a
reduction in the expression of the proneural gene ac in the
proneural cluster corresponding to the affected macrochaetae.
Third, weak but generalised ectopic expression of either sal or
salr results in ectopic macrochaetae, and in considerable
reorganisation of the corresponding proneural clusters, which
is aggravated by higher expression levels of salr. Interestingly,
ectopic expression of wg in the thorax represses or leads to the
misplacement of a set of macrochaetae that are mostly
unaffected by overexpression of Sal/Salr (Fig. 7C versus E,F).
Many macrochaetae, corresponding to proneural clusters and
SOPs that are included in the domain of sal/salr expression,
differentiate normally in the absence of these genes. This
indicates that at these positions sal/salr are not essential for
regulation of proneural genes, possibly because other activators
of proneural genes can provide a redundant function. In
summary, our results place Sal/Salr in the group of transcription
factors implicated in the spatial activation of the proneural
genes. This group also includes the proteins encoded by the
iroquois gene complex (Gómez-Skarmeta et al., 1996) and
Pannier/U-shaped (Cubadda et al., 1997). These transcription
factors are expressed in a complex landscape of overlapping
domains in the thorax, which could be part of the postulated
prepattern that directs SOPs positioning (Stern, 1954). It is not
clear whether these overlapping expression territories are
generated independently of each other, or whether, alternatively,
some cross-regulatory interactions between these transcription
factors participate in their refinement.
In contrast to its earlier expression throughout the pertinent
proneural clusters, we find that Sal disappears from the SOPs
from the moment that SOPs can be identified using the
enhancer trap neu-lacZ. The elimination of Sal from the SOP
and its progeny is of critical importance for differentiation of
these cells, because strong ectopic expression of either sal or
salr in the SOPs prevents or delays their differential divisions
and leads to the disappearance of most of the macrochaetae or
their replacement by very small bristles. SOP cells express a
characteristic class of genes named pan-neural, such as asense
(Dominguez and Campuzano, 1993) and deadpan (Bier et al.,
1992), which participate in SOP differentiation and confer on
2662 J. F. de Celis, R. Barrio and F. C. Kafatos
them specific characteristics. It is likely that future experiments
might reveal that sal/salr interferes with the expression or
activity of some of the pan-neural genes, resulting in the failure
of SOP differentiation.
There are some parallels between the role of sal/salr in the
patterning of veins and sensory organs in the wing and thorax,
respectively. In both cases, the expression of these genes is
under the regulation of signalling molecules (Dpp and
Wg/Hh/Dpp respectively) and occurs in restricted domains. In
both processes, sal and salr participate in the positioning of
pattern elements, and they may do so by determining the places
where cell differentiation promoting genes are expressed. The
identification of sal/salr regulators and target genes is therefore
of critical importance for deepening our understanding of the
development of the wing disc, and for revealing the molecular
mechanisms that link the activity of signalling molecules such
as Dpp, Wg and Hh with the final pattern of differentiation.
We thank M. Ashburner, in whose laboratory part of this work has
been carried out, I. Rodriguez, K. Basler, M. Dominguez, S. Cohen,
A. Martinez-Arias, J.L. Mullor, S. Campuzano, R. Schuh, A.
Martinez-Arias and M. Noll for reagents and fly stocks. We are
grateful to A. Carpenter for help in generating the sal/salr mutants
and to S. Bolshakov for help in characterising them. We also thank
B. Miñana for technical assistence and A. M. Voie for germline
transformations. J. F. dC. is supported with a Wellcome Trust grant
and R. B. with a postdoctoral fellowship from the Spanish Ministerio
de Educación y Cultura.
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