Download A Directed Mutagenesis Screen in Drosophila

Copyright  2000 by the Genetics Society of America
A Directed Mutagenesis Screen in Drosophila melanogaster Reveals
New Mutants That Influence hedgehog Signaling
Nicola Haines and Marcel van den Heuvel
MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom
Manuscript received June 16, 2000
Accepted for publication August 15, 2000
ABSTRACT
The Hedgehog signaling pathway has been recognized as essential for patterning processes in development of metazoan animal species. The signaling pathway is, however, not entirely understood. To start
to address this problem, we set out to isolate new mutations that influence Hedgehog signaling. We
performed a mutagenesis screen for mutations that dominantly suppress Hedgehog overexpression phenotypes in the Drosophila melanogaster wing. We isolated four mutations that influence Hedgehog signaling.
These were analyzed in the amenable wing system using genetic and molecular techniques. One of these
four mutations affects the stability of the Hedgehog expression domain boundary, also known as the
organizer in the developing wing. Another mutation affects a possible Hedgehog autoregulation mechanism, which stabilizes the same boundary.
M
EMBERS of the Hedgehog (Hh) family of proteins are secreted intercellular signaling molecules that provide vital patterning information in a wide
range of developmental contexts. Signaling by Hh proteins has been identified in patterning of the vertebrate
neural tube, the somites, specification of different neuronal cells, and myotome and sclerotome differentiation. The determination of left-right asymmetry, hair
follicle development, and limb morphogenesis have all
been found to involve Hh signaling (for review, see
Hammerschmidt et al. 1997). The hh mutation was first
identified in a mutagenesis screen for embryonic lethals
in Drosophila melanogaster that disrupt the segmental organization of the embryo (Nusslein-Volhard and
Wieschaus 1980). Strong hh alleles cause deletion of
the posterior naked portion of each embryonic segment
and loss of cell polarity in the remaining (denticle-covered) part, resulting in a “spiky” embryo with deranged
denticles everywhere. This phenotype led to the hh mutant being classed as one of the segment polarity mutants. This group of mutants is now well characterized
and the underlying genes are known to be required for
correct patterning and polarity of each embryonic trunk
segment (Martinez-Arias et al. 1988). The hh gene was
cloned (Mohler and Vani 1992) and its amino acid
sequence suggested that the protein would be secreted.
Within the segment polarity group of mutants, several
lead to phenotypes similar or identical to the hh mutant
phenotype. It is now clear that some of these genes are
required downstream of the Hh signal (Ingham 1998).
Corresponding author: Marcel van den Heuvel, MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University
of Oxford, S. Parks Rd., Oxford OX1 3QX, United Kingdom.
E-mail: [email protected]
Genetics 156: 1777–1785 (December 2000)
Hh genes were cloned from vertebrate species on the
basis of homology to the Drosophila hh gene (see Hammerschmidt et al. 1997). In addition, for most of the
genes identified in Drosophila as functioning in the
Hh pathway, homologous vertebrate genes have been
isolated. At least for some of these, a direct role downstream of vertebrate Hh proteins has been defined similar to what is seen in flies. Loss of hh function in mice
is embryonic lethal and midline defects are seen in
humans in whom the hh gene is affected (Chiang et
al. 1996; Roessler et al. 1996). A driving role for Hh
signaling in the generation of oncogenic transformation
has been described on the basis of mutations in Hh
signaling genes (see Ruiz i Altaba 1999). The bestknown example of this is the role the patched (ptc) gene
plays in the noninvasive skin tumors of the basal cell
carcinoma type (Johnson et al. 1996). The ptc gene
normally functions to inhibit Hh signaling. This becomes obvious as the ptc loss-of-function phenotype in
Drosophila embryos is identical to the phenotype
caused by overexpression of hh (Ingham 1993). In fact,
ptc encodes the only protein yet known to bind the Hh
protein directly (Stone et al. 1996).
As in vertebrates where Hh proteins play important
roles in the development and differentiation of many
tissues, the single hh gene in flies (based on latest genomic database searches) plays multiple roles. Its expression in the embryonic segment is maintained in the
tissues that will form most of the adult fly. In these
imaginal discs, hh plays an essential role in patterning
(Basler and Struhl 1994).
Although several proteins are known to function
downstream of Hh, either to transduce or to inhibit
the signal (Ingham 1998), their interactions are poorly
understood. To identify possible new components in or
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N. Haines and M. van den Heuvel
modifiers of Hh signaling, we made use of the role Hh
signaling plays in the patterning of the imaginal wing
disc in Drosophila. An existing dominant hh allele,
hhMoonrat, induces overgrowth and repatterning in the
wing (Tabata and Kornberg 1994). The hhMrt phenotype is mild and thus can be used as a background to
isolate suppressors. Several mutants were isolated that
reduce the phenotype and these are presented here
with their genetic analysis.
MATERIALS AND METHODS
Fly strains: A second chromosome isogenized stock was generated (cn bw sp) for the mutagenesis. All GAL4 lines (30A,
34B, and 1348) have been described (Brand and Perrimon
1993). The following deficiency stocks were used: Df(2L)J39,
Df(2R) PC4, Df(2R)or-BR-11, and Df(3R)vin7; some of these
form part of the Bloomington deficiency kits. UAS hh, UAS
sonic hh, UAS dpp, UAS ci, and dpplacZ have been described
(Ingham and Fietz 1995; Alexandre et al. 1996). The stocks
used for meiotic recombination mapping were net dp cn bw,
net bw sp, dp cn bw, and st p e.
Mutagenesis: A total of 10,000 male flies of an isogenized
cn bw sp stock were treated with 4000 rad using a Co source.
After 24 hr they were mated to hhMrt/TM3 females and the F1
non-TM3 progeny were screened for reduction of the hhMrt
phenotype. The suppressing loci were rescued, tested again
over hhMrt, and balanced using the suppression phenotype to
determine the chromosomal location. All stocks were then
kept balanced or as homozygous (if viable). Regularly, the
suppression phenotype was tested against either hhMrt or 30AGAL4 UAS shh.
␤-Galactosidase stainings of imaginal discs: Larval heads
were cut off the larvae in 1⫻ PBS. They were inverted and
fixed for 6 min in 1⫻ PBS, 0.1% glutaraldehyde. The inverted
heads were washed five times with 1⫻ PBS and incubated at
37⬚ for 1 hr in 10 mm phosphate buffer, 3.1 mm K4(Fe[II]
CN6), 3.1 mm K3(Fe[III]CN6), 150 mm NaCl, 1.0 mm MgCl2
with 0.32% X-gal (5-bromo-4-chloro-3-indolyl ␤-d-galactopyranoside). The heads were washed with 1⫻ PBS, 0.1% Tween
20, and the discs were dissected in the same buffer. The wing
imaginal discs were mounted in 1⫻ PBS, 70% glycerol on
glass slides. They were photographed with a Zeiss Axioscop
microscope using differential interference contrast optics.
Antibody stainings of imaginal discs: Larval heads were cut
off the larvae in 1⫻ PBS on ice. They were inverted and fixed
for 20 min in 1⫻ PBS, 4% paraformaldehyde. The inverted
heads were washed five times with 1⫻ PBS and again washed
five times for 5 min each with 1⫻ PBS, 5% normal serum
(usually donkey in case secondary antibodies were derived
from donkey), and 0.3% saponin (PBT). They were incubated
overnight at 4⬚ with the primary antibodies in PBT and washed
again five times with PBT. Secondary antibodies were obtained
from Jackson Immunoresearch Laboratories (West Grove,
PA). They were used at the manufacturer’s recommended
concentrations for 2 hr in PBT at room temperature, after
which the heads were washed again five times with PBT. Discs
were dissected from the heads in PBT and mounted in VectaShield (Vector Laboratories, Burlingame, CA) on glass slides.
They were viewed using a Leica confocal laser scanning microscope.
The antibody against the Cubitus interruptus (Ci) protein
originated from Motzny and Holmgren (1995), while the
antibody against Engrailed (En) protein came from Patel et
al. (1989).
RESULTS
Suppression of hhMrt phenotype: The hhMrt allele leads
to ectopic expression of the hh gene in the anterior
wing compartment, where normally hh is not expressed
(Tabata and Kornberg 1994; Felsenfeld and Kennison 1995). The resulting phenotype, shown in Figure
1B, varies between clear overgrowth of the (usually distal) part of the wing to slight disorganization of the
wing margin and the addition of extra vein material.
This phenotype can be used to screen for suppressing
mutations; however, its variability can raise difficulties.
Male flies were exposed to 4000 rad of radiation.
These males were mated to hhMrt females and the resulting F1 progeny were studied. In total, 25,000 chromosomes were screened and 12 mutants were identified in
which the hhMrt phenotype was suppressed. Seven of the
mutations were stabilized and balanced to second and
third chromosomes [labeled Su(hh) I–VII]. These consistently suppressed the hhMrt wing phenotype. To compensate for the isolation of spurious suppressors due to
the variability of the hhMrt phenotype, all isolated mutants
were screened three times against the hhMrt stock; average
suppressed wings are shown in Figure 1, C–I.
Figure 1.—D. melanogaster wings showing the suppression
of hedgehogMrt phenotypes; distal to the left and anterior upward. Wings were derived from adult flies of the following
genotypes: (A) ⫹/⫹; (B) hhMrt/⫹; (C) Su(hh) I/⫹; hhMrt/⫹;
(D) Su(hh) II/⫹; hhMrt/⫹; (E) Su(hh) III/⫹; hhMrt/⫹; (F)
Su(hh) IV/hhMrt; (G) Su(hh) V/⫹; hhMrt/⫹; (H) Su(hh) VI/
hhMrt; (I) Su(hh) VII/hhMrt. Note suppression of the hhMrt phenotype in all wings (C–I); wings are representatives of at least
three individual crosses and several hundred flies.
Analysis of hedgehog Signaling
Suppression of ectopic hh phenotypes: In the hhMrt
wing imaginal discs, hh is ectopically expressed in the
anterior region of the disc. To verify that our mutants
did not suppress Hh overexpression specifically in this
region of the wing, all isolated mutants were crossed
into a background where Hh is expressed in the disc
at this and other sites. However, ectopic expression of
Drosophila hh in the wing imaginal disc using transgenes
leads to severe duplications and often flies do not
emerge. Vertebrate Hh genes induce similar but milder
duplications when overexpressed (Figure 2B). In addition, by using a vertebrate Hh gene, we reassert that
the observed suppression is specific to Hh signaling.
The seven mutants were tested using a phenotype
generated by UAS-GAL4-driven expression of zebrafish
sonic hh in the presumptive proximal areas of the wing
(Brand and Perrimon 1993; Krauss et al. 1993). Using
30AGAL4, four of the seven mutants clearly suppressed
the strong wing duplication phenotype caused by overexpression of shh [Figure 2, C–F; Su(hh) I–IV]. Three
mutants did not suppress this phenotype [Figure 2, G–I;
Su(hh) V–VII]. Using a second GAL4 driver (34BGAL4)
that drives expression in cells that will form only the
most proximal parts of the wing, the duplication is
milder (Figure 2J). Su(hh) I–IV that suppress the strong
duplication phenotype driven by 30AGAL4 UAS shh also
suppress the 34BGAL4 phenotype (Figure 2, K–N).
Suppression of ectopic decapentaplegic phenotypes: A
transcriptional target of Hh signaling in the wing imaginal disc is the decapentaplegic (dpp) gene, a member of
the tgf␤/BMP superfamily (Zecca et al. 1995). The Dpp
protein is expressed in a narrow band of cells adjacent
to the hh expression domain (see Figure 5A). The Dpp
protein is considered responsible for some of the growth
and patterning in the wing imaginal disc (Nellen et al.
1996).
The suppression of the overgrowth and repatterning
phenotypes seen above could be caused by suppression
of Dpp signaling rather than of Hh signaling itself. To
distinguish between these possibilities we introduced
the Su(hh) I–VII mutants into a dpp overexpression background. In this experiment, the dpp gene is ectopically
expressed using the same GAL4 driver as above (30A),
leading to a mirror image duplication of tissue at the
anterior and overgrowth at the posterior (Figure 3B).
Suppression of the phenotype caused by dpp overexpression was observed in four mutants [Su(hh) I, II, IV, and
VII; Figure 3, C, D, F, and I). The strong suppression
observed in Su(hh) VII was very consistent, while in
Su(hh) I, II, and IV only mild suppression was observed
and sometimes it was impossible to distinguish suppressed from nonsuppressed. Three mutants did not
suppress the dpp-induced phenotype [Su(hh) III, V, and
VI; Figure 3, E, G, and H].
On the basis of these and the above results, we excluded Su(hh) V and Su(hh) VI (shown in Figures 2 and
3, G and H) from further study; these did not suppress
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Figure 2.—D. melanogaster wings showing the suppression
of sonic hedgehog overexpression phenotypes; distal to the left
and anterior upward. Wings were derived from adult flies of
the following genotypes: (A) ⫹/⫹; (B) 30AGAL4; UAS shh/
⫹; (C) 30AGAL4/Su(hh) I; UAS shh/⫹; (D) 30AGAL4/Su(hh)
II; UAS shh/⫹; (E) 30AGAL4/Su(hh) III; UAS shh/⫹; (F)
30AGAL4/⫹; UAS shh/Su(hh) IV; (G) 30AGAL4/Su(hh) V;
UAS shh/⫹; (H) 30AGAL4/⫹; UAS shh/Su(hh) VI; (I)
30AGAL4/⫹; UAS shh/Su(hh) VII; ( J) 34BGAL4 UAS shh; (K)
34BGAL4 UAS shh/Su(hh) I; (L) 34BGAL4 UAS shh/Su(hh)
II; (M) 34BGAL4 UAS shh/Su(hh) III; (N) 34BGAL4 UAS shh/
⫹; Su(hh) IV/⫹. Note that Su(hh) V–VII do not suppress the
30AGAL4 UAS shh phenotypes.
ectopic shh nor ectopic dpp. Su(hh) VII clearly reduces
the phenotype caused by ectopic dpp but the phenotypes
caused by ectopic hh expression are not affected (Figures 2 and 3I). As we were interested in Hh signaling, we
did not pursue this mutant either. Of the four remaining
mutants, Su(hh) III (Figure 2, E and M; Figure 3E)
suppresses only ectopic hh expression. The other three
[Su(hh) I, II, and IV; Figures 2 and 3, C, D, and F; Figure
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N. Haines and M. van den Heuvel
Figure 3.—D. melanogaster wings showing suppression of
the decapentaplegic overexpression phenotypes; distal to the left
and anterior upward. Wings were derived from adult flies of
the following genotypes: (A) ⫹/⫹; (B) both 30AGAL4/⫹;
UAS dpp/⫹; (C) 30AGAL4/Su(hh) I; UAS dpp/⫹; (D)
30AGAL4/Su(hh) II; UAS dpp/⫹; (E) 30AGAL4/Su(hh) III;
UAS dpp/⫹; (F) 30AGAL4/⫹; UAS dpp/Su(hh) IV; (G)
30AGAL4/Su(hh) V; UAS dpp/⫹; (H) 30AGAL4/⫹; UAS dpp/
Su(hh) VI; (I) 30AGAL4/⫹; UAS dpp/Su(hh) VII.
2, K, L, and N] suppress ectopic hh consistently and
strongly. In addition, these three mutants [Su(hh) I, II,
and IV] suppress the phenotypes caused by ectopic dpp
mildly and sometimes not at all. We believe this variability might indicate that these suppressors act at the level
of dpp transcriptional induction. Alternatively, it indicates that the variable suppression is due to the fact
that ectopic dpp drives developmental changes in the
posterior compartment (and hh using our driver does
not), which might influence the observed phenotypes.
Complementation and mapping analysis: To genetically localize the isolated mutants, we performed meiotic recombination mapping making use of the suppression of the 34BGAL4 UAS shh phenotype. Mapping of
the mutants would allow us to define the mutants as
possible novel genes interacting with the Hh signaling
pathway or as additional alleles of previously characterized mutants in the pathway. The results of meiotic
mapping of Su(hh) I–IV are shown in Figure 4. In addition to the determination of the approximate map position, deficiencies covering the indicated regions were
tested for suppression of the 34BGAL4 UAS shh phenotype and for genetic interactions with the isolated mutants (Figure 4).
Figure 4.—Results of meiotic recombination mapping depicted as regions within chromosome arms; shown are left
and right arm of second and left arm of third chromosome.
Also shown are deficiencies that take out (parts of the) regions
identified in the meiotic mapping. Su(hh) I maps to position
31–33 on the left arm of the second chromosome (hatched
bar). Recombination frequency with net, 46%; with dp, 18%.
A deficiency (Df(2L)J39) within this region (31C–31D) suppressed the GAL4-derived phenotype (gray hatched bar).
Su(hh) II maps to the region 53A–57A on the right arm of the
second chromosome (vertically hatched bar). Recombination
frequency with cn 19%; with bw, 12%. Again a deficiency
(Df(2R)PC4) within this region suppresses the ectopic shh phenotype (gray vertically hatched bar). Su(hh) III is late embryonic lethal. The mutant was mapped to the region 59–60 using
the suppression of the ectopic shh phenotype. Recombination
frequency with bw, 0%. This mutant is lethal over a deficiency
uncovering 59F–60A (Df(2R)br11); this deficiency also suppresses the test phenotypes. Su(hh) IV was mapped to the left
arm of the third chromosome to 68–69 using meiotic mapping
based on suppression. Recombination frequency with st, 9.5%;
with p, 10%. The mutant is lethal over a deficiency with
breakpoints at 68C and 69B (Df(3L)vin7); again this deficiency
also suppresses the test phenotypes.
Su(hh) I was mapped to map position 31–33 and
Su(hh) II to 53–57. Deficiencies that fall within these
regions also suppressed the 34BGAL4 UAS shh phenotype. Neither of these regions contains mutants that
are thought to influence Hh signaling. Su(hh) III was
mapped to the region 59–60 on the right arm of the
second chromosome. A deficiency covering this region
(59F–60A) suppresses the 34BGAL4 UAS shh phenotype. Su(hh) III is homozygous embryonic lethal and
lethality is also seen in trans over the deficiency. Su(hh)
III mutant embryos display a normal cuticle phenotype.
In the 59–60 region on the second chromosome, several
interesting genes are known. One of these is the glass
bottom boat (gbb) gene that encodes a dpp homologue in
flies. Mutations in this gene have been shown to influence Dpp-driven wing patterning, possibly by cooperating with Dpp signaling (Haerry et al. 1998). Our locus,
however, complements gbb.
Su(hh) IV was mapped to map position 68–69 on the
Analysis of hedgehog Signaling
left arm of the third chromosome. A deficiency in this
region (68C–69B) also suppresses the 34BGAL4 UAS
shh phenotype. Like Su(hh) III, Su(hh) IV is homozygous
lethal and no embryonic phenotype is discernible.
Su(hh) IV is also lethal in trans over the 68C–69B deficiency. There are no mutants known in this region that
influence Hh signaling.
Influence on target gene expression: Ectopic Hh signaling leads to induction of ectopic dpp transcription.
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If our suppressor mutants function in the Hh pathway,
they should reduce the amount of ectopic dpp expression. Figure 5A visualizes the expression domain of the
GAL4 driver we used in these experiments (30AGAL4).
If 30AGAL4 is used to express the shh gene, dpp expression is induced in the areas where the driver is active
in the anterior area of the wing imaginal disc, in addition to the normal expression domain in the stripe
(Figure 5, B–E; left vs. middle). In the posterior part
of the disc (where endogenous hh is expressed), ectopic
dpp is suppressed by the action of the transcriptional
repressor En, coexpressed there with hh (Sanicola et
al. 1995).
We introduced Su(hh) I–IV into the 30AGAL4 UAS
shh genetic background and examined the expression
of dpp using a dpplacZ transgene. Both Su(hh) I and IV
reduced the amounts of ectopic dpp expression compared to the transgenic background, as expected (Figure 5, B and E). In Su(hh) III discs, the ectopic expression of dpp is reduced but also the normal endogenous
stripe of dpp expression through the middle of the disc
is reduced in intensity (Figure 5D). In Su(hh) II, the
levels of ectopic dpp were increased especially in areas
where 30AGAL4 expression is highest (Figure 5C).
Suppression of ectopic cubitus interruptus phenotypes:
Transcription of target genes downstream of hh signaling is elaborated through the activation of a large cytoplasmic complex (Kalderon 1997; Aza-Blanc and
Kornberg 1999). This complex consists of at least three
proteins: Costal-2, a kinesin-like molecule; Fused, a serine-threonine kinase; and Cubitus interruptus (ci), a
zinc-finger transcription factor. The Ci protein is believed to enter the nucleus, to bind upstream to the
DNA of Hh transcriptional targets, and to activate transcription. This has been shown in assays in yeast (Alexandre et al. 1996). The cytoplasmic complex is thought
to mediate Ci activation, perhaps through release to
allow entrance into the nucleus but also through proteolytic control.
Overexpression of the full-length ci gene leads to extreme duplication phenotypes and no flies emerge when
the 30AGAL4 driver is used. We used another GAL4
driver to express ci that allows flies to emerge with a mild
Figure 5.—Dissected wing imaginal discs stained for ␤-galactosidase activity, showing the expression of the 30AGAL4 transgene (A) and decapentaplegic-lacZ transgene (B–E); anterior to
the left; wing pouch upward. The imaginal discs were derived
from third instar larvae of the following genotypes: (A)
30AGAL4 UAS lacZ; (B–E) left, dpplacZ/⫹; middle, dpplacZ/
30AGAL4; UAS shh/⫹. Right (B) 30AGAL4 dpplacZ/Su(hh) I;
UAS shh/⫹; (C) 30AGAL4 dpplacZ/Su(hh) II; UAS shh/⫹;
(D) 30AGAL4 dpplacZ/Su(hh) III; UAS shh/⫹; (E) 30AGAL4
dpplacZ/⫹; UAS shh/Su (hh) IV. Note the increase in ectopic
dpplacZ staining in the disc derived from Su(hh) II larvae (arrowheads) and the reduction of the endogenous stripe of dpp
expression in the Su(hh) III disc (arrow). The control discs
(left and middle in B–E) were derived from the same staining
dish as the suppressed examples shown.
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N. Haines and M. van den Heuvel
Figure 6.—Drosophila wings showing the suppression of
phenotypes generated by overexpression of cubitus interruptus
(A–E); distal to the left and anterior upward. Wings were
derived from adults of the following genotypes: (A)
1348GAL4/⫹; UAS ci/⫹; (B) 1348GAL4/Su(hh) I; UAS ci/⫹;
(C) 1348GAL4/Su(hh) II; UAS ci/⫹; (D) 1348GAL4/Su(hh)
III; UAS ci/⫹; (E) 1348GAL4/⫹; UAS ci/Su(hh) IV. Note the
differential suppression of the ci overexpression phenotype in
the two compartments in Su(hh) IV (compartment boundary
indicated by dashed line).
vein phenotype (1348GAL4; Figure 6A). We introduced
the suppressor mutants into this background and analyzed the suppression of the UAS-ci phenotype. Su(hh)
I did not suppress the phenotype caused by overexpression of ci (Figure 6B). Su(hh) II and III reverted the
phenotype to close to wild-type vein patterning (Figure
6, C and D), while Su(hh) IV effected differential rescue
of the phenotype with anterior compartment vein patterning being close to normal (Figure 6E).
Further analysis of Su(hh) II and III: Su(hh) III was
analyzed further because this locus seems to suppress
only Hh signaling and not Dpp signaling. In Su(hh) III/⫹
discs in a shh overexpression background, reduced levels
of dpp expression at the anterior-posterior compartment
boundary are observed (Figure 5D). In wild-type wing
imaginal discs, the expression of dpp is confined to a
precise stripe of cells anterior to the expression domain
of hh, creating a sharp boundary between hh- and dppexpressing cells (Figure 7A). The Hh signaling pathway
is required for the expression of dpp across this boundary but is also thought to play a crucial role in the
maintenance of the boundary. We were interested to
see if markers of this boundary were disrupted in Su(hh)
III heterozygous mutant wing imaginal discs. The expression of dpp along this boundary in anterior cells is
Figure 7.—Dissected wing imaginal discs stained with antibodies against the ␤-galactosidase protein (representing dpp
expression), and Cubitus interruptus and Engrailed proteins
showing the alteration of their expression in Su(hh) III at the
compartment boundary; anterior upward, wing pouch to the
left. The discs were derived from third instar larvae of, left,
cn bw sp/⫹ or, right, Su(hh) III/⫹ genotypes. Note the changes
in dpp, Ci, and En expression domains in the center of the
disc, which will go on to form the central wing area. The arrow
in A points to the loss of dpp expression in the wing pouch
area. Similarly, the arrow in B points to the wing pouch area
in a disc stained for Ci expression.
abnormal; the normal sharp stripe is diffuse and not as
much a “straight” line down the disc as in wild type
(Figure 7A). However, we could only analyze this using
the dpplacZ transgenic line since antibodies to Dpp protein are no longer available. As alternative markers for
the boundary, we analyzed the expression of the Ci and
En proteins in discs using antibodies directed against
the proteins. The levels of the Ci protein are elevated
in cells where the Hh pathway induces its release from
the cytoplasmic complex, but the protein is absent from
cells expressing hh. Lower levels of Ci protein are seen
throughout the rest of the disc where hh is not expressed
(Aza-Blanc et al. 1997). The En protein is coexpressed
Analysis of hedgehog Signaling
with the cells expressing hh (Tabata and Kornberg
1994). As shown in Figure 7, B and C, Ci and En distributions are both disrupted. The stripe of high protein
levels of Ci normally running very sharply along the
boundary now forms a diffuse variable line down the
middle of the disc. Similarly, the expression domain of
the En protein, which in wild type exactly complements
the Ci stripe, is diffuse and disorganized. The area most
affected in the discs is in most cases the more distal part
of the presumptive wing blade area (arrows in Figure 7).
In three of the suppressor mutants analyzed, we detected a reduction in the levels of target gene expression
induced by the overexpression of Hh. However, in
Su(hh) II, although it clearly suppressed the overexpression phenotypes (Figure 2D), higher levels of ectopic
dpp were found especially in the areas of the disc where
the GAL4 expression is highest (Figure 5C). Hh signaling has been shown to lead to expression of En in
anterior cells, but only in cells that are closest to the hh
expression domain (de Celis and Ruiz-Gomez 1995;
Guillen et al. 1995; Tabata et al. 1995; Gomez-Skarmeta and Modolell 1996; Sanchez et al. 1996; Blair
and Ralston 1997; Mullor et al. 1997; Strigini and
Cohen 1997). It has been proposed therefore that it is
the high levels of Hh that these cells are exposed to
that induce the cells to express En. Possibly the high
levels of ectopic shh in our experimental setup could
induce En expression in the anterior part of the disc.
En activity represses dpp expression directly; ectopic expression of En could thus lead to absence of dpp expression. We first investigated if the expression of shh in the
anterior compartment induces ectopic En expression.
In the GAL4 UAS shh background, ectopic En is seen
in the anterior compartment in the areas where the
ectopic shh expression is highest (Figure 8B). Second,
we investigated whether in Su(hh) II discs the induction
of En expression was lost. In Su(hh) II 30AGAL4 UAS
shh discs, En expression in the part of the imaginal disc
was not observed (Figure 8C). This indirectly would
lead to the gain of dpp expression.
DISCUSSION
We present here four mutations that influence Hh
signaling. These have not been previously isolated as
suppressing Hh-induced patterning and they thus represent new genes that might play a role in Hh signaling.
Alternatively, some of these could be acting in pathways
downstream of Hh patterning activity (i.e., the Dpp signaling pathway).
We utilized a known dominant allele of hh (hhMrt) in
an extensive radiation mutagenesis screen. The combination of this hh allele and transgenes to overexpress
vertebrate Hh genes increased the specificity with which
the mutants could be selected for further analysis. Additional combinations of the mutants with transgenic
backgrounds for genes in the Hh signaling pathway
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Figure 8.—Dissected wing imaginal discs stained with antibodies against the ␤-galactosidase and Engrailed proteins
showing the induction and suppression of induction of Engrailed expression; anterior upward, wing pouch to the left.
The discs were derived from third instar larvae of (A) 30AGAL4
dpplacZ/⫹; (B) 30AGAL4 dpplacZ/⫹; UAS shh/⫹; (C)
30AGAL4 dpplacZ/Su(hh) II; UAS shh/⫹. Left, En expression;
right, lacZ expression (representing dpp expression). The
arrow in B points to the site where ectopic En (left) leads to
loss of ectopic dpp expression. Note that this area is where
the highest levels of ectopic shh are induced (compare to
driver expression in Figure 5A). The arrowhead in C points
to the area where now in Su(hh) II the ectopic En expression
is not seen. Note that the antibody against En leads to some
background but this background is not nuclear (nuclei show
up as small dots).
allowed a placement in the genetic hierarchy. In addition, by using vertebrate homologues for Hh overexpression, the possibility of finding evolutionarily conserved
components in the Hh pathway will have increased.
We isolated one mutant, Su(hh) I, that suppressed
the phenotypes caused by overexpression of hh (or shh),
and this mutant suppresses the induction of ectopic
target gene expression (dpp) by Hh signaling. It maps to
the left arm of the second chromosome at map position
31–33. No mutants that affect Hh signaling have been
characterized in this region. This mutant could thus
1784
N. Haines and M. van den Heuvel
represent a new gene that functions in the Hh pathway.
Interestingly, a homologue of the ptc gene can be found
here in the annotated genome database (accession no.
CG5722). This ptc homologue shows significant homology to the human NPC 1 gene; this gene was isolated
in humans as the cause of the Nieman-Pick C1 disease
(Carstea et al. 1997).
In contrast, in Su(hh) II discs, we found that the expression domain of the Hh target gene dpp expands.
This locus was isolated as the strongest suppressor of
Hh overexpression phenotypes; it also suppresses albeit
weakly ci and dpp overexpression phenotypes. Upon further analysis of the induction of dpp in this mutant
background, we found that the gain of dpp in the discs
is due to a loss of ectopically induced En. We show that
ectopic shh induces endogenous En expression in the
anterior compartment (for references see results). In
Su(hh) II discs, En expression is lost in the areas where
high levels of shh ectopically induced it. En suppresses
dpp expression as a transcriptional repressor (Sanicola
et al. 1995), thus loss of En will lead to gain of dpp
expression, as seen in our mutant. However, in the
30AGAL4 UAS shh wing imaginal discs, in addition to
En expression in the cells exposed to high levels of shh,
endogenous hh expression is also seen in these cells
(data not shown). We do not know if this ectopic hh is
directly induced by ectopic shh or dependent on the
ectopic En. The expression of endogenous hh in these
cells does, however, point to a possible change of cell
identity of these cells, from anterior (non-hh-expressing
cells) to posterior (hh-expressing) cells. The interpretation of the loss of dpp expression in these cells might
thus be due to the fact that these cells are now “true”
posterior cells. Our results indicate, though, that the
Su(hh) II acts in a pathway that leads to concomitant
En and hh expression in the anterior compartment induced by Hh signaling. It is thought that the signaling
leading to En expression in the anterior compartment
proceeds through the normal Hh pathway (Blair and
Ralston 1997; Strigini and Cohen 1997; Ohlmeyer
and Kalderon 1998). Since Su(hh) II also suppresses
ectopic ci phenotypes, we would favor a position for
Su(hh) II downstream of ci function but upstream of
the induction of hh and En expression. It would thus
function in a Hh autoregulatory pathway, upstream of
hh and En expression but downstream of the Hh signaling pathway. Su(hh) II maps to the right arm of the
second chromosome at map position 53–57 and no
genes previously characterized as interacting with the
Hh signal transduction pathway are known to map to
this position.
Both Su(hh) III and IV are embryonic lethal but neither shows any distinct abnormal cuticle phenotype.
If the lethality of these mutants is associated with the
suppression and if these mutants are absolutely required
for Hh signaling, perhaps embryonic segmentation phenotypes would be expected. The apparent lack of these
might indicate a maternal contribution for the gene.
This would be consistent with the fact that these loci
have not been found in mutagenesis screens for zygotic
embryonic phenotypes that resemble the hh phenotype.
We have not generated any germline clones to remove
maternal contribution since the use of radiation as a
mutagen might have caused mutations in more than one
gene in our mutants. A role in embryonic patterning for
these mutants is thus possible but one cannot exclude
an exclusive role for these genes in wing/imaginal Hh
patterning.
Su(hh) III maps to a region where several loci of
interest to wing patterning driven by Hh or Dpp signaling have been placed (map position 59–60). We show
that our mutant is not allelic to gbb, a dpp homologue
in this region that has been shown to cooperate with
Dpp signaling in disc patterning. Another gene situated
close by is the G-protein ␣-subunit encoding gene (G␣s).
Since the Hh signaling pathway contains a putative
G-protein-coupled receptor, smoothened (van den Heuvel and Ingham 1996), a G-protein might influence
Hh signaling. However Su(hh) III does not appear to
map to G␣s either, since a combination of a deficiency
covering Su(hh) III and G␣s combined with a translocation containing G␣s to the X chromosome still suppresses the test phenotypes. Upon further analysis of
the expression of dpp in discs heterozygous mutant for
Su(hh) III, we observed differences in the normal expression domain. The normally sharp boundary between the posterior and anterior compartments was less
defined, leading up to (in extreme cases) a very disrupted and unclear compartment boundary. Indeed,
we confirmed this observation using other markers for
the boundary, Ci and En. It is known that Hh signaling
is required for proper establishment of the boundary
but little is known of the cell biology underlying the
clonal and affinity restrictions at the boundary (Blair
and Ralston 1997; Rodriguez and Basler 1997; Dahmann and Basler 2000). It is possible that Su(hh) III
is in some way involved downstream of Hh in establishing the boundary. Consistent with its being downstream
of Hh but upstream of Dpp signaling is that Su(hh) III
suppresses ectopic ci but not ectopic dpp. There are
many loci in this region of the chromosome and further
analysis will be needed to determine which of these
genes (or more) are affected in Su(hh) III.
Su (hh) IV is localized on the third chromosome to
map position 68–69. It suppresses well the phenotypes
generated by overexpression of shh. It also weakly suppresses those generated by dpp ectopic expression. One
observation indicates a particular role for this locus.
When ci is overexpressed to a low level overlying the
boundary between hh-expressing and nonexpressing
cells, rescue of the resulting phenotype is observed but
only in the domain of the wing that did not express hh.
This result indicates that this locus plays a role in Hh
signaling at the level of Ci or directly downstream but
Analysis of hedgehog Signaling
only in anterior compartment cells since here the cidriven phenotypes are rescued. There are no genes in
this area that show homology to genes known to act in
the Hh signaling pathway.
We acknowledge the generous contributions of Drosophila stocks
by the Bloomington Stock Center, Dr. Norbert Perrimon, Dr. Jym
Mohler, Dr. Andrea Brand, Dr. Kristi Wharton, and Dr. David Roberts.
The antibodies against the Ci protein were a gift of Dr. R. Holmgren.
We thank Dr. Philip Ingham for helping us with the original idea to
use the hhMrt stock as the subject of our screen. And we thank Dr.
David Roberts for useful comments on the manuscript. This work
has been made possible by Biotechnology and Biological Sciences
Research Council grant 43/G09234 and a Medical Research Council
program grant to M.v.d.H. as part of the MRC Functional Genetics
Unit.
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Communicating editor: T. Schüpbach