1 Centre de Biologie du Développement-CNRS and Institut d'Exploration
Fonctionnelle du Génome, 118 route de Narbonne, Bâtiment 4R3,
F-31062 Toulouse Cedex 04, France
2 Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel,
Switzerland
3 Department of Biology and Biochemistry, University of Houston, 369 Science and
Research Bldg. 2, Houston TX 77204-5001, USA
4 IBCG-CNRS, Université Paul Sabatier, 118 route de Narbonne, F-31062
Toulouse Cedex, France
* Author for correspondence (e-mail: cribbs{at}cict.fr)
Accepted 15 October 2002
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SUMMARY |
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Key words: Hox, Pax6, proboscipedia, Maxillary palps, Protein-protein, Differentiation
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INTRODUCTION |
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Despite the identification of several target genes, important gaps remain
in our understanding of the pathway from patterned Hox gene expression to the
development of diverse segmental morphologies
(Mann, 1995). For example, the
poor DNA sequence specificity exhibited by Hox transcription factors in vitro
contrasts with their highly specific effects in vivo. One source of enhanced
Hox specificity derives from their joint action with cofactors such as
extradenticle and its mammalian PBX family counterparts that enhance
sequence selectivity and alter in vivo specificity
(Chan et al., 1994
;
Passner et al., 1999
;
Pinsonneault et al., 1997
;
Rauskolb et al., 1993
;
van Dijk and Murre, 1994
). An
important role for stoichiometric combinations of homeotic proteins with
functional partners is supported by observations suggesting that Hox protein
levels can be crucial for their developmental effects
(Cribbs et al., 1995
;
Greer et al., 2000
;
Roch and Akam, 2000
;
Smolik-Utlaut, 1990
). A
combinatorial coding model predicts that different Hox genes and their
partners acting together can specify novel cell or segment identities,
deploying developmental pathways that are different to those specified by one
gene product acting alone.
The Drosophila proboscipedia (pb) gene codes for a
conserved homeodomain protein required for the correct development of the
adult fly mouthparts (Cribbs et al.,
1992) where two distinct appendages, the labial and maxillary
palps, show a dose-sensitive requirement for pb gene function
(Kaufman, 1978
;
Pultz et al., 1988
). These
appendages derive from the labial and antennal imaginal discs, respectively.
Labial palp differentiation requires the combinatorial action of two Hox
genes, pb and Scr
(Percival-Smith et al., 1997
).
In contrast, pb appears to be necessary and sufficient for the
differentiation of maxillary palps, and ectopic PB expression induces a
dose-sensitive transformation of antennae to maxillary palps
(Cribbs et al., 1995
). Another
consequence of ectopic PB expression is a striking dose-sensitive eye loss.
The observation that PB protein incapable of DNA binding can induce this eye
loss suggested that context-specific protein-protein interactions are central
to this defect (Benassayag et al.,
1997
). We attempted to identify specific protein partners of PB by
isolating dominant Enhancer mutations for the PB-dependent eye loss. Four such
mutations are new alleles of the eyeless gene encoding a D.
melanogaster Pax6 homolog.
The Pax gene class encodes proteins with a DNA-binding Paired domain that
may be coupled with a class-specific homeodomain
(Bopp et al., 1986;
Frigerio et al., 1986
;
Gehring et al., 1994
;
Manak and Scott, 1994
;
Strachan and Read, 1994
;
Stuart et al., 1994
). Reduced
Pax6 function in mammals leads to the defective eye structures
typical of the dominant congenital disorder Aniridia in humans
(Ton et al., 1991
), or
Small eye (Sey) in mice
(Hill et al., 1991
). Further
roles for Pax6 in mouse development are revealed in homozygous
mutants lacking nasal structures or showing brain defects
(Glaser et al., 1994
;
Hogan et al., 1988
;
Schmahl et al., 1993
). The
spectrum of abnormalities seen in Sey mice thus indicates that
Pax6 is required in eye, nose and brain development. Loss-of-function
eyeless mutations in Drosophila lead to reduced or defective
eye structures, whereas targeted expression of EY protein induces ectopic eyes
(Halder et al., 1995
;
Quiring et al., 1994
). Recent
work has also revealed that apart from this central role in forming the
Drosophila eye, eyeless is also required for brain
development (Callaerts et al.,
2001
; Kurusu et al.,
2000
; Noveen et al.,
2000
).
To better understand the roles of the eye selector gene eyeless (ey) and the basis of its functional relationship with proboscipedia (pb) in normal development, we analyzed the molecular structures and the mutant phenotypes of the four newly identified eyeless mutations. These alleles code for truncated EY proteins and are expected to affect all aspects of ey function. The mutant phenotypes indicate that eyeless is required for adult maxillary palp differentiation. Furthermore, both EY and PB accumulate in the maxillary primordium beginning early in pupal development. We show that PB-induced eye loss occurs via post-translational repression of EY-mediated target gene transcription. Furthermore, we detect direct protein-protein binding in vitro involving the PB homeodomain and the EY Paired domain. Direct protein-protein binding in a modified yeast two-hybrid analysis is likewise sufficient to diminish or abolish EY transcriptional activation from a target promoter, sine oculis. These genetic and molecular results support a direct functional antagonism between the Hox gene pb and the Pax gene ey in maxillary palp development and provide evidence for a functional link integrating eyeless with the position-specific Hox gene network.
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MATERIALS AND METHODS |
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Fly strains
All stocks and crosses were maintained at 25°C on standard
yeast-agar-cornmeal medium. Specific fly lines or alleles used were:
soPlacZ (Cheyette et
al., 1994), dpp-Gal4
(Staehling-Hampton et al.,
1994
), UAS-ey (Halder
et al., 1995
), ey-GAL4
(Halder et al., 1998
),
UAS-pb 49.1 (Aplin and Kaufman,
1997
), HSPBsy and wt
(Benassayag et al., 1997
;
Cribbs et al., 1995
),
pbGAL4 UASLacZ/CyO (constructed by L. Joulia). The eya and
so alleles eyaclift1, eya2,
so1 and soL31 were from L. Pignoni and L.
Zipursky; eygM3-12 and eygc1 were from
Henry Sun; ey2 and eyR were as
described (Quiring et al.,
1994
); and Df(4)BA was provided by E. Frei and M. Noll
(Kronhamn et al., 2002
).
Specific genotypes generated for this publication were: UAS-pb;
UAS-ey and solacZ/CyO; dpp-GAL4/TM6B,
Hu Tb.
Phenotypic analysis
Detailed phenotypic analysis was by light microscopy (Zeiss Axiophot) after
mounting dissected samples in Hoyer's medium, or by scanning electron
microscopy (SEM). For SEM analysis, adult flies were stored in 95% ethanol
until dissection of heads. Preparation and mounting were as described
(Benassayag et al., 1997).
Molecular characterization of eyeless mutations
The molecular characterization of eyeless mutations involved PCR
amplification of all the exons from intronic primers, and sequencing of the
cloned products. For a detailed description, see Callaerts et al.
(Callaerts et al., 2001).
Histology
In situ hybridizations on larvae or on prepupae were performed using
digoxigenin-labeled probes according to Sturtevant and Bier (as described at
http://www-bier.ucsd.edu/imagdisc.html).
Antibody stainings were performed according to Halder et al.
(Halder et al., 1998) at the
following dilutions: rabbit
ey (1/500), rabbit
pbE9 (1/175),
mouse
eya (1/50), mouse
dac (1/200) and mouse
ß-gal (1/5000).
Pull down experiments
GST fusion proteins were produced and purified according to the
manufacturer's specifications (Pharmacia). Quantification of the GST fusion
protein was monitored by Coomassie staining to ensure equivalent amounts of
GST fusion proteins in the assay. 35S-labeled proteins were
synthesized using a coupled in vitro transcription translation kit (TNT
Promega). The binding conditions were modified from Chen et al. (Chen et al.,
1997): Glutathione resin-bound GST proteins were pelleted and washed three
times with 500 µl of binding buffer (20 mM Hepes, 1 mM DTT, 0.1 mM EDTA,
2.5 mM MgCl2, 150 mM NaCl, 0.05% (v/v) NP40, 10% (v/v) glycerol, 100 µg/ml
BSA). Labeled proteins were incubated in 0.4 ml binding buffer with
approximately 10 µg of bound GST fusion proteins for 2 hours at 4°C.
The resin was washed five times with 1 ml of binding buffer, then bound
35S proteins were eluted by boiling for 3 minutes in 25 µl of
loading buffer, fractionated by SDS-PAGE and visualized by autoradiography
(from 15 to 36 hours). We measured bound labeled proteins eluted compared with
the input by quantitation on a Phospho-imager.
`One and a half hybrid' experiments
One-hybrid experiments were performed using the MATCHMAKER one hybrid
system (Clontech) according to the manufacturer's specifications. The
NcoI-SmaI fragment of ey cDNA was subcloned in
frame with the Gal4DB in the pAS vector. The PB fragment (amino acids 128-305)
was fused to the GAL4 transactivation domain in the pACT vector. pAS and pACT
are from Durfee et al. (Durfee et al.,
1993).
Plasmid constructions
Plasmids used for GST fusion protein production: pEn24-4
(Bourbon et al., 1995) leads
to GST-EN fusion protein (EN-374/552) containing HD and adjacent regions.
PLS15: pGEXB PBµ4a produces a GST-PB isoform µ4a (amino acids 126-306)
containing HD and adjacent regions
(Seroude and Cribbs, 1994
).
PLS20: pGEXB PBsy produces the same GST-PB protein carrying the
point mutation in the HD transforming Arg5 to His
(Benassayag et al., 1997
). In
vitro transcription or translation was performed using cDNAs of eyeless,
eyes absent, sine oculis and extradenticle cloned respectively
in the following plasmids: pBSN eyeless
(Quiring et al., 1994
), pblue
eyaI (Bonini et al., 1993
),
pBSpF3k, and pSp64 ATGexd (van Dijk and
Murre, 1994
). The truncated EY proteins were generated by deletion
of the full-length eyeless cDNA contained in the pBSN plasmid. To
generate a EY
HD protein, ends generated by a partial HindIII
digestion (position 773) and a complete XbaI digestion of
pBSN-eyeless were blunted by filling in and the plasmid recircularized. This
construct expresses a truncated protein containing the 245 first amino acids,
including the Paired Domain (27 kDa). To obtain the EY
PD protein pBSN
eyeless was digested with NcoI (made blunt by filling in) and
BamH1 (made blunt by exonuclease treatment) and recircularized. This
construct generates a protein of 681 amino acids (75 kDa) deleted for 157
amino acids (between 18-174). To generate the EY
PB
HD protein,
pBSN eyeless was digested with NcoI (making blunt by filling in) and
NruII and recircularized. It leads to a protein of 387 amino acids
(43 kDa) deleted for 451 amino acids (18- 468).
Construction of a pb-GAL4 driver: GAL4 coding sequences were
placed with pb control elements within 6.2 kilobase pair (kb)
SalI fragment harboring pb exons 1 and 2, intron 1, and
regulatory sequences in intron 2 (Cribbs
et al., 1992; Kapoun and
Kaufman, 1995
). The circular form of the 6.2 kb fragment subcloned
in the plasmid pGEM2 was used for amplification by PCR. One primer, situated
in exon 1, was oriented upstream and contained restriction sites for
BamHI and NotI; the second primer, situated at the 3'
end of exon 2, was oriented downstream and contained restriction sites for
NotI and XbaI. The amplified product of 6 kb was digested
with NotI, ligated and transformed into E. coli. This
plasmid is deleted for the 3' end of exon 1, intron 1 and exon 2. GAL4
coding sequences removed from pGATB (Brand
and Perrimon, 1993
) by digestion with BamHI and
NotI were inserted into the pb plasmid cut with the same
enzymes. Finally, an EcoRI/SalI fragment containing
pb exon 1 as a transcriptional fusion with GAL4 coding sequences,
plus known intron 2 regulatory elements, was inserted into CaSpeR4 cut with
EcoRI and XhoI. This pb-GAL4 plasmid was injected
into flies together with a plasmid supplying transposase, and transgenic lines
established by standard methods. The properties of these lines will be
described in detail elsewhere (L. Joulia, H. M. Bourbon and D. L. C.,
unpublished).
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RESULTS |
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In a search for such functional protein partners of pb also
involved in head morphogenesis, we screened for dominant mutations enhancing
the observed eye defect. We employed the mutant transgene HSPBsy,
because PBsy protein retains the capacity of wild-type PB to induce
eye loss but is less toxic for flies. Males hemizygous for the X-linked
HSPBsy element are normal (Fig.
1A), and homozygous females (two transgene copies) are nearly so
(Fig. 1B). In contrast,
complete eye loss occurs in females carrying four copies
(Fig. 1C). HSPBsy
males were treated with the chemical mutagens EMS or DEB and crossed with
homozygous females (two copies of HSPBsy). We then selected
candidate female progeny (two copies) with a strong eye loss resembling four
copies (Fig. 1C). Among
approximately 80,000 F1 HSPBsy/HSPBsy females screened,
approximately 20 new Enhancer mutations were retained. These mutations
represent at least five loci, distributed on all four chromosomes. Three of
the Enhancer mutations were new alleles of Notch
(Boube et al., 1998). Four
En(sy) alleles mapped to the fourth chromosome and were identified as alleles
of the eye development gene eyeless based on complementation analyses
with three previously characterized eyeless alleles,
ey2, eyR and Df(4)BA
(Kronhamn et al., 2002
;
Quiring et al., 1994
).
Specific genetic antagonism between pb and ey in
PB-induced eye loss
The strength of the new ey alleles (eyJD,
eyEH, ey11 and eyD1Da) was
compared with ey2 and Df(4)BA for its interaction with
HSPBsy (Fig. 1D). On
crossing HSPBsy/Y; ey*/+ males with
HSPBsy/HSPBsy females, half the female progeny should be
HSPBsy/HSPBsy; ey*/+ and eye loss
could attain at most 50%. In this test, sensitized females heterozygous for
the eye-null allele ey2 showed 12% eye loss; similarly,
heterozygotes for the recently described deletion Df(4)BA
(Kronhamn et al., 2002) showed
13% eye loss. In contrast, most of the new alleles showed a more marked
interaction. eyJD, eyD1Da and
eyEH interacted similarly with HSPBsy
(Fig. 1D; eye loss between 25
and 29%), although ey11 was markedly weaker (6%). These
results suggesting that the eyJD, eyEH and
eyD1Da alleles are stronger than ey11
are in general agreement with the results of complementation tests employing
ey2, eyR and Df(4)BA
(Callaerts et al., 2001
) (data
not shown). Thus three of the new ey alleles interact more strongly
with HSPBsy than the described null alleles. To examine the
specificity of the observed eye loss, we tested for genetic interactions with
sine oculis (so), eyes absent (eya) and
eye gone (eyg) (Bonini et
al., 1993
; Cheyette et al.,
1994
; Jun et al.,
1998
), prominent players in the eye differentiation cascade
(Fig. 1D)
(Halder et al., 1998
;
Niimi et al., 1999
;
Zimmerman et al., 2000
). None
showed a marked interaction with PBsy
(Fig. 1D). The remaining
unidentified Enhancer loci from our screen complement mutants of all three
genes, and thus do not appear to correspond to characterized members of the
eye hierarchy. In conclusion, these results support a specific genetic
antagonism between pb and eyeless/Pax6 in programmed eye
loss.
The new eyeless alleles encode truncated proteins
The eyeless locus encodes a transcription factor with two highly
conserved DNA-binding motifs, a paired domain (PD) and a homeodomain (HD)
(Callaerts et al., 1997). Two
of the best characterized eyeless alleles, ey2
and eyR, result from insertion of mobile elements within
eye-specific regulatory sequences (Quiring
et al., 1994
). In contrast, the four new eyeless alleles
isolated by their interaction with PB all affect the EY protein itself, based
on the molecular lesions identified by PCR amplification and sequencing of
genomic exons (Callaerts et al.,
2001
) (Fig. 1E).
Sequence analysis revealed that eyJD (EMS-induced) harbors
a point mutation and encodes a protein truncated in the homeodomain after the
first helix. The DEB-induced eyD1Da removes the C-terminal
156 amino acids (Fig. 1E),
whereas for a second DEB-induced allele, eyEH, a 6 bp
sequence in exon 4 (positions 423-428 in exon 4) is replaced by a single T
nucleotide (Fig. 1F). The
resulting frameshift should yield a truncated protein of 115 amino acids
deleted at the C-terminal end of the Paired domain
(Fig. 1E).
ey11 allele harbors an EMS-induced point mutation in the
splice acceptor site of intron 8 (Fig.
1F; CAG
CAA). Translation of unspliced mRNA retaining intron
8 is expected to terminate at a stop codon immediately after the exon 8,
resulting in a protein truncated by 199 amino acid
(Fig. 1E). This molecular
characterization thus confirmed the identity of these Enhancer mutations as
ey alleles that should affect the EY protein itself. In the case of
eyJD and eyD1Da this point has been
validated (Callaerts et al.,
2001
).
eyeless function is required for adult maxillary and
antennal development
To better understand the relationship between PB and EY in normal
development, we examined the phenotypic effects of ey mutations in
the sensitized HSPBsy genetic context. Two copies of the
HSPBsy transgene (the sensitizing condition) showed no marked
effect (Fig. 1B). In contrast,
pharate adult females with 2x HSPBsy and homozygous for
eyJD showed strong maxillary palp and antennal defects
(Fig. 2B,C). In some cases, the
maxillary palp, whose identity is indicated by the distinctive distal
bristles, remains adjoined to the antennal appendage
(Fig. 2C, arrowheads). However,
differentiation of the proboscis (which likewise depends on pb
function) is not affected (Fig.
2B, `lb'). Thus in the sensitized context, ey mutations
can provoke strong defects of the maxillary and antennal appendages. The
phenotype of Fig. 2B in which
the appendages are poorly resolved, suggests that ey+ may
participate in partitioning imaginal disc cells into antenna and maxillary
palp during morphogenesis.
|
To confirm a role of eyeless in this process, we removed HSPBsy from the genetic background to examine the effects of the new eyeless mutations alone. All four ey alleles appear recessive in a non-sensitized background as shown for eyJD (Fig. 2A,D), and can be interpreted as loss-of-function mutations in accord with their molecular lesions. All give homozygous escapers with visible defects, allowing us to compose an allelic series, from weakest to strongest: ey11>eyD1Da>eyEH>eyJD. Analysis of the phenotypes of hemizygotes with Df(4)BA led to the same conclusion.
eyJD homozygotes display eye reduction or loss
(Fig. 2G), low viability and
strong brain defects associated with abnormal behavior
(Callaerts et al., 2001).
Furthermore, a minority of surviving eyJD homozygotes
(10-20%, after outcrossing) show alterations in the size and/or shape of
maxillary palps and antennae (Fig.
2G-I). The altered maxillary palps of eyJD
homozygotes still harbor the two characteristic sensilla trichodea
(Fig. 2H, arrowheads),
suggesting that maxillary identity per se is not affected. Reduced, malformed
maxillary palps are often accompanied by enlarged, misshapen antennae (compare
Fig. 2E and 2H; 2F and 2I). Similar although weaker defects were likewise detected for
eyEH homozygotes, as well as for certain
trans-heterozygous combinations with other Enhancer alleles in the sensitized
background (not shown). The reciprocal effect of eyJD on
appendages that derive from the same antennal imaginal disc constitutes
evidence for a potential role for ey in apportioning the maxillary
portion of this disc. The defects observed in eyJD
homozygotes appeared stronger than in the hemizygous combination,
eyJD/Df(4)BA. Thus, the truncated protein may have a
limited antimorphic character not detected in the presence of wild-type
protein.
EY and PB proteins are both expressed in the pupal maxillary
primordium
Although mutant phenotypes implicated both pb and now ey
in maxillary development, no gene expression had been detected in the
maxillary portion of the antennal disc in the third instar larvae. We analyzed
PB and EY expression later, during the prepupal stage when maxillary and
antennal structures evaginate from the eye-antenna imaginal disc
(Fig. 3A). Using a rabbit
anti-PB serum directed against the C-terminal region (anti-E9), PB
accumulation was detected in the central part of the maxillary primordium
beginning approximately eight hours after puparium formation, during
evagination of the antennal and maxillary appendages from the composite disc
(Fig. 3B). EY protein as
visualized by a rabbit anti-EY serum accumulates in the same primordium and,
within discrimination, at the same time
(Fig. 3C). This expression
appeared to be limited to the borders of the primordium
(Fig. 3C, higher
magnification), rather than the center as for PB
(Fig. 3B, higher
magnification). Results of tests for co-expression of the two proteins were
mitigated: in situ hybridization or immunostaining experiments were
inconclusive, whereas available antibodies that gave acceptable signals in
this tissue were both rabbit polyclonal antisera. To address whether
endogenous pb and ey patterns in the maxillary palps may
overlap, we utilized a pb-GAL4 mini-gene based on previous
descriptions of the pb-promoter region
(Kapoun and Kaufman, 1995).
Using a pb-GAL4 driver insertion to direct ß-galactosidase
expression (pb-GAL4>UAS-lacZ), we examined the patterns
of pb>lacZ and ey expression by double-immunofluorescence
labeling and confocal microscopy. Early PB expression is limited to a small
number of cells in the distal maxillary primordium
(Fig. 3D and 3F, green). At
this stage, EY expression could likewise be detected in a small group of cells
partially overlapping those expressing PB
(Fig. 3F, yellow arrowheads).
Co-expression appears to be very limited in the progression of a dynamic
pattern (compare Fig. 3F and
3G). In later prepupae, the expression patterns of
pb>lacZ and ey in the maxillary primordium are adjacent
but exclusive (Fig. 3G). Taken
together, these data show a previously undisclosed co-temporal expression of
both pb and ey in the maxillary primordium of prepupae, and
support an ephemeral co-expression of these genes in a small number of cells.
This is in agreement with the known function of pb in maxillary
determination, and with the newly established function of ey in this
tissue.
|
PB regulates EY transcription factor activity
To further characterize the molecular basis of the functional relationship
between the two genes, the epistatic relations of pb and ey
were tested. eyeless mRNA accumulates normally in
pb- embryos, third instar larvae or pre-pupae, whereas PB
protein accumulated normally in ey mutants (data not shown). These
observations argued against a transcriptional cross-regulation effected by the
nuclear EY and PB transcription factors.
The mechanism of PB-induced eye loss was examined employing
UAS/GAL4-mediated expression. The eye loss is temporally specific, because
drivers expressed early during eye differentiation (ey-GAL4,
dpp-GAL4) led to substantial or full eye loss
(Fig. 4A; compare parts 1 and
6), whereas the later-acting GMR-GAL4 driver had little effect (not shown). We
next examined the effects of PB on the expression of several identified genes
acting early in the eye cascade (see Fig.
4A). Despite a drastic reduction of the L3 eye disc, early PB
eye-expression (dpp-GAL4>UAS-PB) does not eliminate ey
mRNA accumulation there (Fig.
4A, part 7). In contrast, expression of the ey target
genes so, eya and dac was strongly reduced or abolished
(Fig. 4A, parts 8,9,10,
respectively). One possible explanation is that PB represses the same ensemble
of targets activated by EY. If so, removing pb activity in labial
discs could lead to expression of these eye genes there. Indeed, novel
dac expression is detected in the labial discs of
pb- larvae in accord with the appearance of ectopic legs
(Abzhanov et al., 2001). In
contrast, no corresponding expression was detected for eya or
so in mutant labial discs (compared with endogenous expression in the
eye). These results thus raise an alternative possibility, namely that PB
simultaneously suppresses expression of all three EY targets by antagonizing
ey activity. This could occur via a post-transcriptional mechanism,
acting at the level of EY protein (model,
Fig. 4B).
|
To test whether PB represses EY transcription factor activity, the GAL4
system was used to direct expression of these proteins, singly or together, in
several imaginal discs under a common driver (dpp-GAL4). EY activity
was monitored by formation of adult eyes, and by the expression of the known
target gene sine oculis (so) (employing an enhancer trap
line that fully recapitulates normal so expression in the eye
imaginal disc (Cheyette et al.,
1994). Targeted PB expression induces eye loss, abnormalities in
legs, antennae and wings (see Fig.
4C, part 1) (Aplin and Kaufman,
1997
), and diminished so expression in the eye imaginal
disc (Fig. 4C, parts 1,2).
Targeted expression of EY in the same tissues induces supernumerary eyes
(Fig. 4C, part 4) and
so-lacZ expression in several tissues including the antennal and wing
imaginal discs (arrows, Fig.
4C, parts 5,6). On co-expressing EY and PB, both proteins
co-accumulate, cell by cell, as detected by double immunofluorescence labeling
and confocal microscopy (not shown). In contrast, ectopic eye formation was
strongly reduced or abolished (Fig.
4C, part 7) as was expression of the eye reporter gene so
(compare Fig. 4C, parts 8,9 and
parts 5,6). Normal eye formation and so-lacZ expression were
partially restored on co-expressing PB with increased EY (transgenic plus
endogenous copies), suggesting a stoichiometric relationship for these
proteins. This observation is supported by results obtained with other GAL4
driver lines (not shown).
In vitro protein binding is mediated by the EY Paired domain and the
PB homeodomain
To ask whether PB protein interacts directly with EY, we first tested for
stable in vitro binding in `pull-down' experiments. Glutathione S-transferase
(GST) protein was produced intact, or in chimeric fusion forms containing
homeodomain sequences from PB or ENGRAILED (EN)
(Bourbon et al., 1995). The
GST-PB fusion protein harbored a 180 amino acid interval
(Fig. 5A) containing the
homeodomain, adjacent YPWM motif and a conserved C-motif of unknown function
(Cribbs et al., 1992
) (also
see Materials and Methods). These GST-containing proteins were bound to
glutathione-Sepharose affinity beads, then tested for binding to
35S-labelled probe proteins generated by in vitro transcription and
translation. No binding was detected between immobilized GST protein and any
of the probe proteins tested (EXD, EY, SO and EYA)
(Fig. 5B, lanes 5-8). In
contrast, GST-EN fusion protein effectively bound the homeodomain-containing
EXD protein (Fig. 5B, lane 13)
(van Dijk and Murre, 1994
),
but not the homeodomain-containing EY and SO nor the nuclear protein EYA
(Fig. 5B, lanes 14,15,16). On
testing with the same probes, efficient binding of GST-PB to EXD was observed
(Fig. 5B, lane 9), as predicted
based on the YPWM motif present in PB and implicated in binding to the
homeotic co-factor EXD (Johnson et al.,
1995
). On testing the three proteins of the eye cascade, PB
binding was detected with EY (Fig.
5B, lane 12), but not with EYA or SO
(Fig. 5B, lanes 10,11). These
data support direct, specific contacts between EY and the PB
homeodomain-containing region.
|
The EY protein contains two DNA-binding domains, a paired domain (PD) and a
homeodomain (HD) (see Fig. 1E).
Of these, the PD has been shown to interact with other transcription factors
(Bendall et al., 1999;
Fitzsimmons et al., 1996
;
Jun and Desplan, 1996
;
Plaza et al., 1997
;
Plaza et al., 2001
). To define
sequences within EY required for interaction with PB, we tested deleted forms
of EY lacking the PD, the HD, or the region containing both, for their ability
to bind the GST-PB fusion protein. EY deleted of its HD retains PB-binding
capacity comparable to the intact protein
(Fig. 5C, lane 6). In contrast,
EY protein deleted for the PD (
PD), with or without the HD and linker,
showed little or no interaction with PB
(Fig. 5C lanes 7,8). This
finding supports a crucial role for the PD in binding with PB.
A PB protein entirely deficient for in vitro DNA binding but harboring the
Arg5His mutation can still induce eye reduction, suggesting that this
point mutation might favor specific protein contacts with some
context-specific eye protein compared to wild-type PB HD
(Benassayag et al., 1997
). The
GST-PB and GST-PBsy proteins were therefore compared for their
relative binding affinities to full-length or deleted forms of
35S-EY as described above. EY protein binding to PB or
PBsy is strongly reduced or undetectable for all forms of EY
lacking a PD (Fig. 5C, lanes
7,8,11,12). In contrast, EY forms containing the PD (complete, or deleted for
the linker and HD:
HD), were bound by PB and PBsy
(Fig. 5C, lanes 5,6). Binding
of input 35S-EY protein to GST-PBsy was consistently
enhanced approximately four-fold compared with GST-PB (see
Fig. 5C, compare lanes 5 and
9). A similar difference was obtained on probing with 35S-EY PD
(
HD, lacking the HD and linker; Fig.
5C, compare lanes 6 and 10). These results, coherent with the
behavior of PB and PBsy in vivo, support a direct role for the N
terminus of the PB HD in binding the EY PD.
PB inhibits EY-dependent transcriptional activation in yeast
To address whether this protein-protein interaction can contribute to the
inhibition of EY activity in a cell, a `one-and-a-half' hybrid assay in yeast
was performed to test the effects of PB on EY-mediated activation of a
sine oculis (so) transcription enhancer. The reporter gene
harbors the so10 fragment of 400 bp, an eye-specific element directly
regulated by EY (Niimi et al.,
1999), upstream of the lacZ reporter. EY expressed from
the pAS plasmid activates so10-lacZ
(Fig. 5D lane 2). PB sequences
were fused to the GAL4 trans-activation domain in pACT-PB, using the same PB
HD-containing peptide as for the pull-down experiments (amino acids 126-306).
The so10 fragment was not activated by the chimeric GAL4-PB protein
(Fig. 5D, lane 4), suggesting
that the so10 fragment is not directly bound by PB. Although EY activates
so10-lacZ in these conditions, co-expression with GAL4-PB protein
inhibited EY-mediated activation (Fig.
5D, lane 3). This result cannot be explained by a saturation
because of the GAL4 activation domain (`squelching'), because this domain
alone does not affect EY activation (Fig.
5D, lane 2). Thus in yeast cells as in vitro, our data favor a
direct contact with PB that reduces EY-mediated so activation in
yeast, consistent with our observations in vivo. Together, these results
strongly support a direct interaction between conserved domains of PB and EY
that accounts for functional antagonism in the ectopic context of developing
adult eye, and in the normal development of the maxillary palp.
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DISCUSSION |
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ey function in maxillary palp development
The present work has identified a previously unrecognized role for
ey in the development of the maxillary palps and antennae. The
mutation employed for most of our experiments here, eyJD,
behaves as a strong allele affecting viability, formation of the adult eyes
and brain mushroom bodies (Callaerts et
al., 2001), but also of antennal and maxillary differentiation.
Consistent with a late requirement for ey in the antennal disc,
ey expression in the maxillary primordium appears in early stages of
metamorphosis when both eye-antennal discs have fused, and the antennal and
maxillary appendages start to evaginate. After evagination, the maxillary
primordium migrates to join the labial disc in forming the adult mouthparts,
whereas the antennal primordium remains near the eye. When a contiguous
epidermal cell layer has been completed the head sac is abruptly evaginated
under the internal pressure (Jürgens
and Hartenstein, 1993
). Maxillary expression in early stages of
prepupal metamorphosis was limited to the boundary between the maxillary
primordium and the antenna, and ey mutant phenotypes often involved
simultaneously reduced palps and enlarged antennae. These reciprocal effects
are consistent with communicating cell populations, suggesting that
ey may contribute to a partitioning of the antennal disc permitting
the establishment of two separate appendages. Further analysis of this process
will require new maxillary-specific markers permitting the fates of these
cells to be followed.
pb/ey antagonism in maxillary versus antennal
development
In the present work, starting from a dose-sensitive eye loss provoked by
ectopic PB, we identified new ey alleles isolated as eye loss
Enhancers. These mutations reveal a role for ey, and a potential
biological relevance for this Hox-PAX6 interaction, in the development of the
antennal and maxillary sensory palps. ey loss-of-function defects in
the sensory palps are exacerbated by PBsy. The most direct
interpretation of the enhanced ey loss-of-function phenotype with
HSPBsy is that the newly isolated alleles retain a partial function
that can be negated by adequate PB levels. The molecular characterization of
these alleles is consistent with this hypothesis, because all four alleles
should encode truncated proteins that contain most or all of the interacting
PD.
To better understand the in vivo relationship between these two selector genes, we sought to define the effects of double mutants for pb and ey. Although homozygotes for pb- or for the new ey mutants showed viabilities of up to 50% compared with heterozygotes, we have never obtained a double mutant adult for any of the four ey alleles. This result, although suggesting that the double mutant is synthetic lethal, does not offer insight into the tissue(s) implicated in this lethality.
One tissue in which an interaction is clearly indicated from our analysis
is the maxillary palp primordium, where we detected a dynamic expression of EY
and of PB (directly or via the pbGAL4 driver) during pre-pupal development.
Transient early co-expression of PB and EY in pre-pupae was limited to a small
number of cells, whereas later expression appears exclusive. This result can
be rationalized in two ways: first, co-expressing cells might be rapidly
eliminated by apoptosis, through a coordinate gene-activation process
triggered by a Hox-Pax dimer; second, co-expression of the EY and PB
transcription factors could induce a developmental pathway interference
resulting in a G1 cell-cycle arrest, as recently suggested by Jiao et al.
(Jiao et al., 2001). These
possibilities are not fully exclusive. Indeed, one or both mechanisms could
serve to refine the boundaries between antennal and maxillary cell populations
within the antennal disc.
Multiple roles of eyeless in head development
In vertebrates, Pax6 has multiple known or inferred roles in eye,
brain and nasal development (Grindley et
al., 1995; Quinn et al.,
1996
). Apart from the fly eye, several groups have identified an
eyeless function required for development of the mushroom bodies,
neural structures important for olfactory perception and learning
(Callaerts et al., 2001
;
Kurusu et al., 2000
;
Noveen et al., 2000
). Our
study describes a specific role for EY in concert with PB in the maxillary and
antennal appendages that are derived from the antennal disc and constitute the
adult olfactory system. A recent analysis of mutations producing headless
flies revealed a new role for Drosophila Pax6 in head morphogenesis
and thereby suggests a requirement of ey for the development of all
structures derived from eye-antennal discs
(Kronhamn et al., 2002
). A
parallel can be drawn with the work presented here because both cases involve
mutations truncating the EY protein which induces head defects. Interestingly,
because these truncated EY proteins still contain the PD, we cannot exclude
the fact that the phenotypes obtained reflect an allele-specific antimorphic
effect of the PD. Taken together, these results strongly suggest that
eyeless, apart from its known role in eye morphogenesis, may also
play multiple other roles in head formation (notably for brain and olfactory
sensory systems).
The development of olfactory and visual systems has several common features
in Drosophila. Both systems are derived from the composite
eye-antennal imaginal disc. Moreover, both have similar signal transduction
pathways and appear to share regulatory networks
(Carlson, 1996;
Gaines and Carlson, 1995
;
Smith, 1996
). However, when we
examined the expression of ey, so, eya and dac in the
pre-pupal maxillary primordium, only ey expression was detected. This
observation suggests that ey acts there via a distinct combinatorial
code of regulatory genes compared with eye development. One possibility
proposed in this study is that ey activity is modulated by other
co-factors or transcription factors whose activity is likely sensitive to PB.
In this light, it is worth noting that other Enhancer mutations isolated in
our screen also similarly affect maxillary palps, singly or in combination
with ey. It will be of fundamental interest to better understand the
molecular basis for how a single protein might function in multiple, distinct
networks.
HOX-PAX6 interaction as a general regulating mechanism
The ey mutants studied here were identified as dominant enhancers
of pb-induced eye reduction. Consistent with the antagonism observed
in vivo, EY and PB proteins interact directly in vitro, via the EY Paired
domain and the PB homeodomain. This interaction with PB that renders EY unable
to activate its downstream target genes can be extended to other homeotic
genes because Antp, Scr, Ubx, abdA and AbdB repress eye
development while increasing apoptosis in the eye disc, and their protein
products likewise interact in vitro with EY protein
(Plaza et al., 2001). This
suggests a combinatorial interaction of homeodomain-containing proteins (Hox
and Pax) to specify a given body segment
(Plaza et al., 2001
). An
inhibition through physical association has been proposed between Pax6 and
En-1 during eye development in quail
(Plaza et al., 1997
), and
between Pax3 and Msx1 for muscle development in chicken
(Bendall et al., 1999
).
Moreover, a similar inhibitory mechanism involving a Hox protein HD has
recently been reported in vertebrates
(Abramovich et al., 2000
;
Kataoka et al., 2001
); in
contrast, physical interaction with Hox-B1 protein led to increased Pax6
activity in Hela cells (Mikkola et al.,
2000
), raising the possibility that additional context-dependent
partners modulate the action of Hox-Pax combinations to generate functional
diversity. Based on our genetic and molecular data, we propose that variations
on a PD-HD interface can serve to mediate combinatorial or hierarchical
functional relationships among Hox and Pax genes in normal development.
The recent paper of Jiao et al. proposed that ectopic expression of Hox and
Pax (and various other) transcription factors in the eye induces a generic
developmental pathway interference (Jiao
et al., 2001). This cellular process was proposed as a general
surveillance mechanism that eliminates most aberrations in the genetic program
during development and evolution, but is not a consequence of specific protein
collaboration. However, the existence of such a mechanism does not preclude
the possibility of more specific interactions such as we describe. The results
presented here appear to favor a specific role for discrete protein-protein
interactions rather than an indirect interference mechanism. Indeed, (i) by
analysing the residues of PB protein involved in its homeotic function, we
identified a PBsy protein with diminished DNA binding but still
able to inhibit eye development; (ii) using this mutant in a genetic screen to
isolate PB functional partners, we isolated four independent eyeless
mutations, all of them leading to a shortened EY protein; (iii) genetic
interaction tests showed that PBsy-induced eye loss is highly
sensitive to levels of ey function but independent of several other
eye-determining genes including eyg, eya or so; and (iv)
ectopically expressed PB interferes with ey activity in the eye
imaginal disc by inhibiting so and eya activation without
affecting ey transcription or EY accumulation.
In conclusion, our results suggest that a specific Hox/Pax interaction between PB and EY is involved in a normal developmental process defining the boundary between the antenna and maxillary palp. More generally, the formation of such protein couples could afford a sensitive and delicate measure of the balance of Pax6 level, permitting a finely tuned integration to generate distinct transcriptional outputs during development.
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ACKNOWLEDGMENTS |
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![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abramovich, C., Shen, W. F., Pineault, N., Imren, S., Montpetit,
B., Largman, C. and Humphries, R. K. (2000). Functional
cloning and characterization of a novel nonhomeodomain protein that inhibits
the binding of PBX1-HOX complexes to DNA. J. Biol.
Chem. 275,26172
-26177.
Abzhanov, A., Holtzman, S. and Kaufman, T. C.
(2001). The Drosophila proboscis is specified by two Hox
genes, proboscipedia and Sex combs reduced, via repression of leg and antennal
appendage genes. Development
128,2803
-2814.
Aplin, A. C. and Kaufman, T. C. (1997). Homeotic transformation of legs to mouthparts by proboscipedia expression in Drosophila imaginal discs. Mech. Dev. 62, 51-60.[CrossRef][Medline]
Benassayag, C., Seroude, L., Boube, M., Erard, M. and Cribbs, D. L. (1997). A homeodomain point mutation of the Drosophila proboscipedia protein provokes eye loss independently of homeotic function. Mech. Dev. 63,187 -198.[CrossRef][Medline]
Bendall, A. J., Ding, J., Hu, G., Shen, M. M. and Abate-Shen,
C. (1999). Msx1 antagonizes the myogenic activity of Pax3 in
migrating limb muscle precursors. Development
126,4965
-4976.
Bonini, N. M., Leiserson, W. M. and Benzer, S. (1993). The eyes absent gene: genetic control of cell survival and differentiation in the developing Drosophila eye. Cell 72,379 -395.[Medline]
Bopp, D., Burri, M., Baumgartner, S., Frigerio, G. and Noll, M. (1986). Conservation of a large protein domain in the segmentation gene paired and in functionally related genes of Drosophila.Cell 47,1033 -1040.[Medline]
Botas, J. (1993). Control of morphogenesis and differentiation by HOM/Hox genes. Curr. Opin. Cell Biol. 5,1015 -1022.[Medline]
Boube, M., Seroude, L. and Cribbs, D. L. (1998). Homeotic proboscipedia cell identity functions respond to cell signaling pathways along the proximodistal axis. Int. J. Dev. Biol. 42,431 -436.[Medline]
Bourbon, H. M., Martin-Blanco, E., Rosen, D. and Kornberg, T.
B. (1995). Phosphorylation of the Drosophila
engrailed protein at a site outside its homeodomain enhances DNA binding.
J. Biol. Chem. 270,11130
-11139.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Callaerts, P., Halder, G. and Gehring, W. J. (1997). PAX-6 in development and evolution. Annu. Rev. Neurosci. 20,483 -532.[CrossRef][Medline]
Callaerts, P., Leng, S., Clements, J., Benassayag, C., Cribbs, D., Kang, Y. Y., Walldorf, U., Fischbach, K. F. and Strauss, R. (2001). Drosophila Pax-6/eyeless is essential for normal adult brain structure and function. J. Neurobiol. 46, 73-88.[CrossRef][Medline]
Carlson, J. R. (1996). Olfaction in Drosophila: from odor to behavior. Trends Genet. 12,175 -180.[CrossRef][Medline]
Chan, S. K., Jaffe, L., Capovilla, M., Botas, J. and Mann, R. S. (1994). The DNA binding specificity of Ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein. Cell 78,603 -615.[Medline]
Cheyette, B. N., Green, P. J., Martin, K., Garren, H., Hartenstein, V. and Zipursky, S. L. (1994). The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron 12,977 -996.[Medline]
Cribbs, D. L., Benassayag, C., Randazzo, F. M. and Kaufman, T. C. (1995). Levels of homeotic protein function can determine developmental identity: evidence from low-level expression of the Drosophila homeotic gene proboscipedia under Hsp70 control. EMBO J. 14,767 -778.[Abstract]
Cribbs, D. L., Pultz, M. A., Johnson, D., Mazzulla, M. and Kaufman, T. C. (1992). Structural complexity and evolutionary conservation of the Drosophila homeotic gene proboscipedia. EMBO J. 11,1437 -1449.[Abstract]
Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilburn, A. E., Lee, W. H. and Elledge, S. J. (1993). The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555-569.[Abstract]
Fitzsimmons, D., Hodsdon, W., Wheat, W., Maira, S. M., Wasylyk, B. and Hagman, J. (1996). Pax-5 (BSAP) recruits Ets proto-oncogene family proteins to form functional ternary complexes on a B-cell-specific promoter. Genes Dev. 10,2198 -2211.[Abstract]
Frigerio, G., Burri, M., Bopp, D., Baumgartner, S. and Noll, M. (1986). Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell 47,735 -746.[Medline]
Gaines, P. and Carlson, J. R. (1995). The olfactory and visual systems are closely related in Drosophila. Braz.J. Med. Biol. Res. 28,161 -167.
Garcia-Bellido, A. (1975). Genetic control of wing disc development in Drosophila. Ciba Found. Symp.161 -182.
Garcia-Bellido, A. (1977). Homeotic and atavic mutations in insects. Am. Zool. 17,613 -629.
Gehring, W. J., Affolter, M. and Burglin, T. (1994). Homeodomain proteins. Annu. Rev. Biochem. 63,487 -526.[CrossRef][Medline]
Glaser, T., Jepeal, L., Edwards, J. G., Young, S. R., Favor, J. and Maas, R. L. (1994). PAX6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects. Nat. Genet. 7,463 -471.[Medline]
Graba, Y., Aragnol, D. and Pradel, J. (1997). Drosophila Hox complex downstream targets and the function of homeotic genes. BioEssays 19,379 -388.[Medline]
Greer, J. M., Puetz, J., Thomas, K. R. and Capecchi, M. R. (2000). Maintenance of functional equivalence during paralogous Hox gene evolution. Nature 403,661 -665.[CrossRef][Medline]
Grindley, J. C., Davidson, D. R. and Hill, R. E.
(1995). The role of Pax-6 in eye and nasal development.
Development 121,1433
-1442.
Halder, G., Callaerts, P., Flister, S., Walldorf, U., Kloter, U.
and Gehring, W. J. (1998). Eyeless initiates the
expression of both sine oculis and eyes absent during Drosophila
compound eye development. Development
125,2181
-2191.
Halder, G., Callaerts, P. and Gehring, W. J. (1995). New perspectives on eye evolution. Curr. Opin. Genet. Dev. 5,602 -609.[CrossRef][Medline]
Hill, R. E., Favor, J., Hogan, B. L., Ton, C. C., Saunders, G. F., Hanson, I. M., Prosser, J., Jordan, T., Hastie, N. D. and van Heyningen, V. (1991). Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature 354,522 -525.[CrossRef][Medline]
Hogan, B. L., Hirst, E. M., Horsburgh, G. and Hetherington, C. M. (1988). Small eye (Sey): a mouse model for the genetic analysis of craniofacial abnormalities. Development 103 Suppl,115 -119.[Medline]
Jiao, R., Daube, M., Duan, H., Zou, Y., Frei, E. and Noll,
M. (2001). Headless flies generated by developmental pathway
interference. Development
128,3307
-3319.
Johnson, F. B., Parker, E. and Krasnow, M. A. (1995). Extradenticle protein is a selective cofactor for the Drosophila homeotics: role of the homeodomain and YPWM amino acid motif in the interaction. Proc. Natl. Acad. Sci. USA 92,739 -743.[Abstract]
Jun, S. and Desplan, C. (1996). Cooperative
interactions between paired domain and homeodomain.
Development 122,2639
-2650.
Jun, S., Wallen, R. V., Goriely, A., Kalionis, B. and Desplan,
C. (1998). Lune/eye gone, a Pax-like protein, uses a partial
paired domain and a homeodomain for DNA recognition. Proc. Natl.
Acad. Sci. USA 95,13720
-13725.
Jürgens, G. and Hartenstein, V. (1993). The terminal regions of the body pattern. In The Development of Drosophila Melanogaster (ed. M. B. and A. Martinez-Arias), pp. 687-746. Cold Spring Harbor: Laboratory Press.
Kapoun, A. M. and Kaufman, T. C. (1995). A
functional analysis of 5', intronic and promoter regions of the homeotic
gene proboscipedia in Drosophila melanogaster.Development 121,2127
-2141.
Kataoka, K., Yoshitomo-Nakagawa, K., Shioda, S. and Nishizawa,
M. (2001). A set of Hox proteins interact with the Maf
oncoprotein to inhibit its DNA binding, transactivation, and transforming
activities. J. Biol. Chem.
276,819
-826.
Kaufman, T. C. (1978). Cytogenetic analysis of
chromosome 3 in Drosophila melanogaster: isolation and
characterization of four new alleles of the proboscipedia (pb) locus.
Genetics 90,579
-596.
Kronhamn, J., Frei, E., Daube, M., Jiao, R., Shi, Y., Noll, M.
and Rasmuson-Lestander, A. (2002). Headless flies produced by
mutations in the paralogous Pax6 genes eyeless and twin of eyeless.
Development 129,1015
-1026.
Kurusu, M., Nagao, T., Walldorf, U., Flister, S., Gehring, W. J.
and Furukubo-Tokunaga, K. (2000). Genetic control of
development of the mushroom bodies, the associative learning centers in the
Drosophila brain, by the eyeless, twin of eyeless, and Dachshund
genes. Proc. Natl. Acad. Sci. USA
97,2140
-2144.
Lawrence, P. A. and Morata, G. (1994). Homeobox genes: their function in Drosophila segmentation and pattern formation. Cell 78,181 -189.[Medline]
Lewis, E. B. and Bacher, F. (1968). Method of feeding ethyl methane-sulphonate to Drosophila males. Dros. Inf. Serv. 43,193 -194.
Manak, J. R. and Scott, M. P. (1994). A class act: conservation of homeodomain protein functions. Development Suppl., 61-77.
Mann, R. S. (1995). The specificity of homeotic gene function. BioEssays 17,855 -863.[Medline]
McGinnis, W. and Krumlauf, R. (1992). Homeobox genes and axial patterning. Cell 68,283 -302.[Medline]
Mikkola, I. I., Bruun, J. A., Holm, T. and Johansen, T. (2000). Superactivation of Pax6-mediated transactivation from paired domain-binding sites by DNA-independent recruitment of different homeodomain proteins. J. Biol. Chem. 7, 7.
Morata, G. (1993). Homeotic genes of Drosophila. Curr. Opin. Genet. Dev. 3, 606-614.[Medline]
Niimi, T., Seimiya, M., Kloter, U., Flister, S. and Gehring, W.
J. (1999). Direct regulatory interaction of the eyeless
protein with an eye- specific enhancer in the sine oculis gene during eye
induction in Drosophila. Development
126,2253
-2260.
Noveen, A., Daniel, A. and Hartenstein, V.
(2000). Early development of the Drosophila mushroom
body: the roles of eyeless and dachshund. Development
127,3475
-3488.
Passner, J. M., Ryoo, H. D., Shen, L., Mann, R. S. and Aggarwal, A. K. (1999). Structure of a DNA-bound Ultrabithorax-Extradenticle homeodomain complex. Nature 397,714 -719.[CrossRef][Medline]
Percival-Smith, A., Weber, J., Gilfoyle, E. and Wilson, P.
(1997). Genetic characterization of the role of the two HOX
proteins, Proboscipedia and Sex Combs Reduced, in determination of adult
antennal, tarsal, maxillary palp and proboscis identities in Drosophila
melanogaster. Development
124,5049
-5062.
Pinsonneault, J., Florence, B., Vaessin, H. and McGinnis, W.
(1997). A model for extradenticle function as a switch that
changes HOX proteins from repressors to activators. EMBO
J. 16,2032
-2042.
Plaza, S., Langlois, M. C., Turque, N., LeCornet, S., Bailly, M., Begue, A., Quatannens, B., Dozier, C. and Saule, S. (1997). The homeobox-containing Engrailed (En-1) product down-regulates the expression of Pax-6 through a DNA binding-independent mechanism. Cell Growth Differ. 8,1115 -1125.[Abstract]
Plaza, S., Prince, F., Jaeger, J., Kloter, U., Flister, S.,
Benassayag, C., Cribbs, D. and Gehring, W. J. (2001).
Molecular basis for the inhibition of Drosophila eye development by
Antennapedia. EMBO J.
20,802
-811.
Pultz, M. A., Diederich, R. J., Cribbs, D. L. and Kaufman, T. C. (1988). The proboscipedia locus of the Antennapedia complex: a molecular and genetic analysis. Genes Dev. 2, 901-920.[Abstract]
Quinn, J. C., West, J. D. and Hill, R. E. (1996). Multiple functions for Pax6 in mouse eye and nasal development. Genes Dev. 10,435 -446.[Abstract]
Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science 265,785 -789.[Medline]
Rauskolb, C., Peifer, M. and Wieschaus, E. (1993). Extradenticle, a regulator of homeotic gene activity, is a homolog of the homeobox-containing human proto-oncogene pbx1. Cell 74,1101 -1112.[Medline]
Roch, F. and Akam, M. (2000). Ultrabithorax and
the control of cell morphology in Drosophila halteres.
Development 127,97
-107.
Schmahl, W., Knoedlseder, M., Favor, J. and Davidson, D. (1993). Defects of neuronal migration and the pathogenesis of cortical malformations are associated with Small eye (Sey) in the mouse, a point mutation at the Pax-6-locus. Acta Neuropathol. 86,126 -135.[Medline]
Seroude, L. and Cribbs, D. L. (1994). Differential effects of detergents on enzyme and DNA-binding activities of a glutathione S-transferase-homeodomain fusion protein. Nucleic Acids Res. 22,4356 -4357.[Medline]
Smith, D. P. (1996). Olfactory mechanisms in Drosophila melanogaster. Curr. Opin. Neurobiol. 6, 500-505.[CrossRef][Medline]
Smolik-Utlaut, S. M. (1990). Dosage
requirements of Ultrabithorax and bithoraxoid in the determination of segment
identity in Drosophila melanogaster. Genetics
124,357
-366.
Staehling-Hampton, K., Jackson, P. D., Clark, M. J., Brand, A. H. and Hoffmann, F. M. (1994). Specificity of bone morphogenetic protein-related factors: cell fate and gene expression changes in Drosophila embryos induced by decapentaplegic but not 60A. Cell Growth Differ. 5,585 -593.[Abstract]
Strachan, T. and Read, A. P. (1994). PAX genes. Curr. Opin. Genet. Dev. 4, 427-438.[Medline]
Stuart, E. T., Kioussi, C. and Gruss, P. (1994). Mammalian Pax genes. Annu. Rev. Genet. 28,219 -236.[CrossRef][Medline]
Ton, C. C., Hirvonen, H., Miwa, H., Weil, M. M., Monaghan, P., Jordan, T., van Heyningen, V., Hastie, N. D., Meijers-Heijboer, H., Drechsler, M. et al. (1991). Positional cloning and characterization of a paired box- and homeobox- containing gene from the aniridia region. Cell 67,1059 -1074.[Medline]
van Dijk, M. A. and Murre, C. (1994). Extradenticle raises the DNA binding specificity of homeotic selector gene products. Cell 78,617 -624.[Medline]
Zimmerman, J. E., Bui, Q. T., Liu, H. and Bonini, N. M.
(2000). Molecular genetic analysis of Drosophila eyes
absent mutants reveals an eye enhancer element.
Genetics 154,237
-246.