1 School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG,
UK
2 Department of Molecular Genetics, 484 West 12th Avenue, The Ohio State
University, Columbus, OH 43210, USA
3 Institute of Child Health, University College, London, 30 Guilford Street,
London WC1N 1EH, UK
Authors for correspondence (e-mail:
bafp8{at}biols.susx.ac.uk
or
simcox.1{at}osu.edu)
Accepted 12 November 2002
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SUMMARY |
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Key words: Drosophila, Imaginal wing disc, Microarray, wingless
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INTRODUCTION |
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Identifying genes has largely relied on the isolation of mutants with
pattern defects, but strategies that detect genes by their expression
patterns, enhancer and protein trapping using P elements with reporter genes
(Bellen et al., 1989;
Bier et al., 1989
), or a GFP
tag (Morin et al., 2001
), have
also identified genes involved in tissue-specific differentiation. However,
P-elements show specificity in their insertion sites
(Liao et al., 2000
), so that
screening the entire genome in this way may remain incomplete.
The annotated genome sequence (Adams et
al., 2000) offers a systematic way to investigate spatial patterns
of expression of all genes in the imaginal discs. Because the predicted gene
number is approximately 14,000, this seems a large undertaking, but is being
done for embryos that lend themselves more readily to high throughput in situ
hybridization
(http://www.fruitfly.org/cgi-bin/ex/insitu.pl).
To identify a subset of genes that may have important roles in wing disc
development, we used hybridization to high-density DNA-oligonucleotide arrays
to define genes that show enriched spatial expression patterns. Cluster
analysis of gene expression throughout the Drosophila life cycle has
led to the identification of muscle-specific transcripts
(Arbeitman et al., 2002
) and
genes expressed in specific imaginal discs, including the wing disc, have been
discovered by profiling individual discs
(Klebes et al., 2002
). Here we
compared RNA profiles from two complementary wing disc fragments, the
presumptive wing/hinge and presumptive body wall regions
(Fig. 1). These regions are
separated by a lineage restriction that occurs in the first larval instar and
genes preferentially expressed in one region may be important for that
fate.
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Our analysis identified many genes with uncharacterized roles in development that have striking spatial expression domains. We discuss the results in the light of the sequence information available for these genes and the role of known genes expressed in similar patterns. Some expression patterns suggest regulation by key morphogens. We have shown that three genes, with robust expression in the wing pouch, are sensitive to wingless (wg) signaling. This work makes a significant contribution to the goal of finding all the genes involved in wing disc development, by identifying a collection of genes that have not been implicated previously but which have expression patterns suggestive of potential region-specific roles.
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MATERIALS AND METHODS |
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Data analysis
Six arrays were used and each demonstrated control parameters within
recommended limits (Raw Q<=30, background <=100, GAPDH 3'/5'
ratios below four). To allow comparison between chips, each was analyzed using
global scaling with a target intensity of 300. The scaling factors used to
normalize to the target value were within four-fold of each other in all
comparisons (range 0.36 to 1.34). Affymetrix Microarray Suite version 5.0
software was used to make each pairwise comparison between the three
wing/hinge and the three body wall arrays. The data were then exported to
Excel, in which the `Signal Log Ratios' were converted to fold changes. Only
transcripts called as present in at least two samples, and showing a two-fold
or greater difference (P0.95) between the wing/hinge and body
wall samples in at least six out of the nine comparisons are included. The
data were sorted by average fold-change to produce the lists of genes shown in
Tables 1 and
2. This filtering of the data
means that some genes with spatially restricted patterns may be excluded; this
is especially likely to be the case for genes with low expression levels.
Therefore, the Excel spreadsheet with the full comparison set is given at
http://dev.biologists.org/supplemental/,
and the array data have been deposited into the Gene Expression Omnibus (GEO)
database
(http://www.ncbi.nlm.nih.gov/geo/)
(series accession number GSE93 and sample accession numbers GSM2583, GSM2584,
GSM2585, GSM2586, GSM2587 and GSM2588).
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cDNA clones and genomic exon fragments used to generate probes for in
situ hybridization
When possible, DIG-labeled RNA probes were generated from cDNA clones
obtained from the Drosophila Gene Collection (DGC,
http://www.fruitfly.org/DGC/index.html)
or from published sources. Most clones used were from the DGC1 release. DGC
clones belonging to the unreleased DGC2 set were ordered from the
Drosophila EST collections maintained by ResGen (Invitrogen). Clones
used are listed in descending order according to the ranked lists (Tables
1 and
2), with the DGC clone
identification number or literature citation as appropriate. Clones were
linearized in the 5' multiple cloning site (except where noted) and
transcribed with the appropriate RNA polymerase.
Notum-enriched list
pOT2a-tailup (GH12431), pOT2a-CG11100 (SD10763), pFlc-CG15064 (RE70039),
pFlc-CG15353 (RH63135), pBS-CG6921 (LD14839), pFlc-BM40/SPARC (RH45818),
pFlc-Obp56a/CG11797 (RE46170), pOT2a-zfh1 (SD06902), pFlc-viking (RE68619),
pBS-Ef1alpha100E (RE68984), pFlc-Obp99a/CG18111 (RH70762), pOT2a-CG10126
(GH22994), pOT2a-CG9338 (GH07967), pBS-Cg25C (GM04010) (this clone failed to
grow and a PCR product was generated, see below), pBS-Act57B (LD04994)
(linearized with AflIII to generate a 3'UTR probe specific to Actin57B
that does not cross-hybridize with other Actins), pOT2a-CG5397 (GH04232),
pFlc-Idgf4 (RE30918), pOT2a-CG4386 (LD47230), pFlc-CG3244 (RH18728),
pFlc-CG2663 (RE73641), pOT2a-CG10275 (LD31354), pOT2a-CG8689/CG30359
(GH18222), pOT2a-tsp/CG11326 (GH27479), pOT2a-Gs2 (GH14412), pFlc-Gld
(RE20037).
Wing/hinge-enriched list
pOT2a-CG17278 (SD04019), pBS-pdm2
(Poole, 1995), pFlc-CG8780
(RE33994), pOT2a-Nep1/5894 (GH03315), pBS-opa
(Benedyk et al., 1994
),
pOT2a-Cyp310a1 (LD44491), pFlc-CG14534 (RE71854), pOT2a-CG9008/BG:DS00797.2
(GH14910), pOT2a-zfh2 (GH11902,), pFlc-Doc2/CG5187 (RE40937) (linearized with
AvaII to produce a specific 3'-end probe that does not cross-hybridize
with Doc1), pOT2a-ana (GH07389), pOT2a-CG8381/CG30069 (LP06813), pOT2a-CG8483
(LD39025), pCR-TOPOII-dorsocross (Lo and
Frasch, 2001
) (linearized with AflII to produce a specific
3'-end probe that does not cross-hybridize with Doc2/CG5187),
pFlc-wengen/CG6531(RE29502).
For genes without cDNA clones available, gene-specific fragments were generated by PCR from genomic DNA using either Taq polymerase (Invitrogen) or Pfu Turbo polymerase (Stratagene). Genomic DNA was generated from adult Canton-S flies using the Qiagen DNeasy Kit. Primers were chosen that would specifically amplify exonic sequence from the gene. The primers were designed using the annotated gene information from GadFly, FlyBase and NCBI/GenBank. Exon numbers mentioned below refer to exon predictions or experimental information from the above sources. Exon fragments were cloned into pBS-KS and verified by sequencing. All clones have a 3' T7 promoter. The fragments generated, the corresponding gene region and the primers used are listed below (written 5' to 3' with any 5'/3' restriction sites introduced underlined) in the order that they appear on the wing/hinge enriched list (Table 2):
Fly stocks
The following transgenic lines were used: 71B-GAL4
(Brand and Perrimon, 1993),
C96-GAL4 (Gustafson and
Boulianne, 1996
), UAS-wg and UAS-DNdTCF
(van de Wetering et al.,
1997
). Larvae from crosses were raised at 29°C to enhance GAL4
activity and produce more extreme phenotypes.
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RESULTS AND DISCUSSION |
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Genes with known restricted expression patterns show enrichment on
the arrays
The rank order of transcripts correlates well with the spatial expression
patterns of characterized genes. In the body wall, pannier
(pan), twist (twi) and BarH1, which are
enriched in the body-wall sample (Table
1), are known to be highly expressed in the presumptive body wall
(Bate et al., 1991;
Ramain et al., 1993
;
Sato et al., 1999
). In the
wing, knot (kn), nubbin (nub) and
Distal-less (Dll) are expressed at levels greater than
10-fold above those in the body wall (Table
2). kn is expressed in the wing 3/4 intervein and hinge
regions (Mohler et al., 2000
;
Vervoort et al., 1999
),
nub is strongly expressed in the entire wing pouch
(Ng et al., 1995
) and
Dll is expressed along the dorsal-ventral (DV) margin exclusively in
the wing pouch (Campbell et al.,
1993
; Gorfinkiel et al.,
1997
).
Other genes, known to have important roles in disc development, appear
lower down the rank order (Table
2). vestigial (vg), a key gene for development
of the wing and hinge regions (Williams et
al., 1991), shows only two-fold enrichment but this is consistent
with the expression pattern of vg in the wing disc that extends into
the body wall region (Williams et al.,
1991
). Transcripts with expression patterns restricted to the
posterior compartment, engrailed (en), invected
(inv) and hedgehog (hh)
(Coleman et al., 1987
;
Kornberg et al., 1985
;
Tabata et al., 1992
), showed
approximately two-fold enrichment in the wing/hinge sample
(Table 2). The
anterior-posterior compartment boundary splits the wing/hinge region into two
equally sized compartments, but the position of the boundary in the body wall
region produces a small posterior compartment representing approximately
one-quarter of the total tissue (Fig.
1B). This is consistent with the approximately two-fold enrichment
of posterior-specific transcripts found in the wing/hinge tissue sample. The
E(spl)-Complex genes are expressed in developing sensory organs found
in both the body wall and wing margin regions. Hence, these genes are not
enriched in any one sample (see
http://dev.biologists.org/supplemental/).
The m6 gene is an exception (enriched in the body wall sample)
(Table 1) and is known to be
expressed only in the body wall region
(Wurmbach et al., 1999
). In
contrast, genes that show ubiquitous expression such as Ras or tubulin show no
enrichment on the arrays (see
http://dev.biologists.org/supplemental/).
Microarray analysis can therefore identify transcripts known to be differentially expressed in the wing/hinge and body wall regions of the disc. Further, the rank order of these by fold change reflects the level of enrichment so that transcripts with more restricted domains appear higher on the list. Few expression patterns of the genes on our list have been described, so to verify the validity of the approach, and to discover more genes with potential roles in the development of these specific regions, we made in situ hybridizations for some of these uncharacterized genes.
In situ hybridization confirms the restricted expression of
previously uncharacterized genes
We analyzed 50 transcripts, shown in Tables
1 and
2, that had strong enrichment
(mostly three-fold or greater). For the body wall-enriched transcripts, the
larger set, we analyzed only transcripts for which clones are available in the
Drosophila gene collections (DGC1 and DGC2, Berkeley Drosophila
Genome Project). For the wing/hinge region, we examined transcripts with
three-fold or greater enrichment, systematically in rank order from the top,
and generated PCR probes when clones were not available. We found that all
transcripts tested showed expression patterns that were consistent with the
microarray data providing confirmation that the microarray analysis mirrors
the spatial distribution of transcripts in vivo. Body wall-specific expression
patterns are shown in Fig. 2
and wing/hinge-specific patterns are shown in
Fig. 3.
Genes with elevated expression in the body wall
The wing disc comprises three cell layers; the squamous epithelium of the
peripodial membrane, the columnar epithelium that becomes the adult epidermis,
and the adepithelial layer that includes myoblast cells that give rise to
adult thoracic muscles and tracheal cells that form air passages
(Fig. 1C). The adepithelial
layer extends from the proximal disc dorsally into the hinge region
(Fig. 1C). The body wall
fragment includes cells of all three layers, so the arrays also identified
transcripts specific to muscle and tracheal cells.
pan and BarH1, which encode transcription factors, are
expressed in the body wall epidermis and are involved in bristle patterning
(Ramain et al., 1993;
Sato et al., 1999
). Both
transcripts were highly enriched on the arrays
(Table 1). Also highly enriched
was tailup (tup) (Thor
and Thomas, 1997
), which encodes a LIM domain homeobox protein,
and is expressed in the epithelium in a large region of the posterior body
wall encompassing the presumptive postnotum, scutellum and scutum
(Fig. 2A). No role for
tup in patterning the mesothorax has been described. Another
transcript with broad expression was thrombospondin/CG11326 (tsp),
which is expressed in a similar region of the body wall to tup
(Fig. 2W). tsp is also
expressed in the ventral hinge and hence shows lower enrichment on the arrays.
The other genes found to be specific to the epithelium showed highly localized
expression: Obp56a/CG11797 (Fig.
2G), CG10126 (Fig.
2L), CG3244 (Fig.
2S) and Glucose dehydrogenase
(Fig. 2Y). Obp56a/CG11797 encodes an odorant-binding protein and interestingly
three other odorant-binding proteins showed enrichment on the arrays
(Table 1): Obp99a/CG18111 (Fig.
2K), CG9358 and Obp56d/CG1128. We found
Idgf4, encoding an imaginal disc growth factor
(Kawamura et al., 1999
), is
expressed in the peripodial membrane, primarily in dorsal cells
(Fig. 2Q). Presumably secretion
of Idgf4 could influence development of the columnar epithelium.
Myoblast cells of the adepithelial layer develop into the direct and
indirect flight muscles of the thorax, and genes involved in the development
of these muscles have been shown to be expressed in the myoblasts during wing
disc development. Several of these transcripts are enriched on the arrays
(Table 1): Mef2
(Cripps et al., 1998),
twist (twi) (Bate et al.,
1991
) and heartless (htl)
(Cripps et al., 1998
).
Act57B is known to be regulated by Mef2 in the embryo
(Kelly et al., 2002
), and we
show Act57B is expressed in the myoblasts
(Fig. 2O), suggesting this
relationship also exists in these adult muscle precursors. Mef2
expression is activated by twi
(Cripps et al., 1998
) and may
be inhibited by the transcriptional repressor, zinc finger homology 1
(zfh1) (Postigo et al.,
1999
). zfh1 is expressed in the myoblasts
(Fig. 2H). stumps is
also enriched on the arrays (Table
1) and expressed in the myoblasts
(Sato and Kornberg, 2002
).
Together with htl, stumps has a role in the development of the
tracheal cells (Imam et al.,
1999
; Sato and Kornberg,
2002
) (see also below). Viking (Vkg) encodes a
component of collagen type IV and is known to be coexpressed with
Cg25C, another collagen IV subunit in the embryo and in blood cells
(Yasothornsrikul et al.,
1997
). Both transcripts are enriched on the arrays
(Table 1) and show similar
expression patterns in the adepithelial myoblasts and blood cells
(Fig. 2I,N). Other genes
showing specific expression in the myoblasts are BM-40/SPARC, a
calcium-binding glycoprotein, which is expressed in the embryonic mesoderm
(Furlong et al., 2001
;
Martinek et al., 2002
)
(Fig. 2F), Elongation
factor 1 alpha 100E (Ef1 alpha)
(Hovemann et al., 1988
)
(Fig. 2J), CG8689, an
alpha-amylase (Fig. 2V), and
two transcripts encoding predicted proteins with unknown function
CG11100 (Fig. 2B) and
CG15064 (Fig. 2C).
In the wing disc, cells of the larval and developing adult tracheal systems
require activity of genes in the FGF pathway
(Sato and Kornberg, 2002).
Some of the key genes are expressed in the myoblasts (for example,
htl and stumps), others in the epithelium (for example,
branchless, bnl), and others in the tracheal cells themselves (for
example, breathless, btl) (Sato
and Kornberg, 2002
). htl and stumps showed
enrichment on the arrays but bnl and btl were not
detectable. For bnl this may be because expression is highly
localized and apparently at very low levels
(Sato and Kornberg, 2002
).
However, it is not clear why the arrays failed to detect btl
expression because we did identify six genes that are also expressed
specifically in tracheal cells. These are CG5397, an
O-acyltransferase (Fig. 2P),
CG4386, a serine-type endopeptidase
(Fig. 2R), CG2663, an
alpha-tocopherol transfer-like protein
(Fig. 2T), and CG15353
(Fig. 2D), CG6921
(Fig. 2E) and CG9338
(Fig. 2M) that have no known
homologies. In particular, CG4386 is interesting as it is only
expressed in the dorsal branch (Fig.
2R), and CG6921 is distinguished because it is very
strongly expressed in the most proximal cells
(Fig. 2E).
Genes with elevated expression in the wing pouch and hinge
regions
The wing/hinge fragment of the wing disc primarily contains cells of the
peripodial membrane and the columnar epithelium
(Fig. 1C), with only a few
myoblasts that extend into the hinge region. Thus the genes detected by the
arrays as enriched in this disc fragment were expressed in cells of one of the
two epithelial layers.
Transcription factors comprise the largest category of genes (18/56) with
elevated expression in the wing/hinge region. These are expected to have
regulatory roles in patterning the region. Transcription factors with known
expression domains and roles in wing development are present: kn, pox-n,
nub, Dll, bifid/optomotor blind, rotund, ventral veins lacking, en, vg
and in (Awasaki and Kimura,
2001; Cohen et al.,
1989
; Coleman et al.,
1987
; de Celis et al.,
1995
; Grimm and Pflugfelder,
1996
; Kornberg et al.,
1985
; Mohler et al.,
2000
; Ng et al.,
1995
; St Pierre et al.,
2002
; Tabata et al.,
1992
; Vervoort et al.,
1999
). pdm2, which is highly related to nub,
also shows wing-enriched expression on the arrays and is expressed in a
similar domain to nub (Fig.
3C) (Ng et al.,
1995
). pdm2 apparently has no significant function in the
wing (Yeo et al., 1995
). The
roles of the remaining seven predicted transcription factors is unknown,
although the expression pattern of zinc finger homology 2
(zfh2) and Sox 15 have been described and both are expressed
specifically in the hinge region (Cremazy
et al., 2001
; Klebes et al.,
2002
). defective proventriculous (dve), which
encodes a homeodomain protein (Nakagoshi
et al., 1998
), and CG15000, which is similar to
NGFI-A-binding protein 2, are broadly expressed in the wing pouch, although
dve is downregulated at the DV compartment boundary
(Fig. 3B,L). odd
paired (opa), known for a role in embryonic segmentation
(Benedyk et al., 1994
), is
discretely expressed in cells of the presumptive mesopleura and dorsal hinge
(Fig. 3G). No role for
opa in wing disc development has been reported. Dorsocross1
(Doc1) (Lo and Frasch,
2001
) and Doc2/CG5187 are T-Box related factors that are
expressed in what appears to be an identical domain in the wing disc
(Fig. 3R,X). Both transcripts
also accumulate in body wall cells and this probably lowers their position in
the overall ranked list (Table
2).
Eight transcripts encoding enzymes are enriched two-fold or greater in the
wing/hinge region (Table 2).
This group includes the most highly enriched transcript detected in the
analysis, a kazal-type serpin gene CG17278 (68-fold,
Table 2). CG17278
shows a strong and specific expression pattern in the wing encompassing most
of the wing pouch. One of the potentially most interesting wing-enriched
enzymes is a cytochrome P450 gene, Cyp310al. This gene is strongly
expressed in the dorsal and ventral parts of the wing pouch but excluded from
the DV and AP boundaries (Fig.
3J). Variable expression in anterior body wall cells is also
observed which is consistent with the array data that indicate
Cyp310al transcripts are also present in body wall RNA. The role of
cytochrome P450 genes in development has recently been reviewed
(Stoilov, 2001), and the list
of these genes with roles in development is growing. Surprisingly, the
ß-galactosidase gene (CG3132) was found to be enriched in the
wing/hinge region (Table 2). ß-gal expression in Drosophila has been analyzed and although it
is expressed in some discs, wing expression was not reported
(Schnetzer and Tyler, 1996
).
We did find weak expression in a cluster of cells in the hinge but the
majority of expression is in blood cells, which adhere preferentially to the
distal disc margin (Fig. 3E).
Thus the ß-gal transcript probably appears as wing/hinge enriched
primarily because it is expressed in blood cells. We also determined the
expression pattern of two other enzymes; the metalloendopeptidase
Nep1/CG5894 (Fig. 3F)
and UDP-glucosyl transferase (Ugt86Di)
(Fig. 3S).
The -integrin, inflated, which has a role in cell adhesion,
is expressed in the ventral compartment
(Brower et al., 1984
) and is
thus enriched on the wing/hinge arrays
(Fig. 1,
Table 2). A novel gene,
CG5758, is potentially involved in cell adhesion as it encodes a
predicted protein with ß-Ig-H3/Fas domains and its expression is
restricted to the dorsal hinge (Fig.
3U). CG8381 encodes a proline-rich protein with repeated
`PEVK' motifs also found in titin. This gene is strongly expressed in the wing
pouch but repressed in cells of the future veins and cells at the DV margin
(Fig. 3V). Despite intense
expression in the wing pouch, CG8381 shows only modest enrichment on
the arrays (Table 2), probably
reflecting the fact that the gene is also expressed in several groups of cells
in the body wall region (Fig.
3V).
The expression of two receptors was determined. CG4861 encodes an
ldl-receptor-like protein and is expressed at very low levels throughout the
wing pouch (Fig. 3O).
wengen/CG6531, which is a receptor of the TNFR family
(Kanda et al., 2002), is
expressed strongly in the wing pouch and weakly in the body wall
(Fig. 3Y). On the arrays, its
ligand, eiger (Kanda et al.,
2002
), was undetectable in the wing/hinge region sample but
enriched in the body wall sample (Table
1).
Two structural proteins, CG6469, a larval cuticle protein, and CG14301, a chitin-binding protein, are the only genes we identified as being expressed in the ventral peripodial membrane. CG6469 is expressed broadly in the peripodial membrane but at a higher level in the ventral region (Fig. 3P). CG14301 is expressed in cells of both epithelial layers, in the columnar epithelium at the anterior disc margin and in four patches of cells in the wing pouch and the overlying peripodial membrane (Fig. 3Q).
In a group of genes with miscellaneous functions
(Table 2) we determined the
expression of three genes. anachronism (ana), a secreted
glycoprotein (Ebens et al.,
1993), is expressed in five clusters of cells including one in the
body wall region and in some individual neuroblasts
(Fig. 3T). ana null
mutants are viable and have no observable defects suggesting it is not
required, or functions redundantly, in the wing
(Park et al., 1997
).
CG14534, which has a domain that has been recognized in several
proteins but has an unknown function (DUF243), is expressed only in cells that
will give rise to the posterior wing margin
(Fig. 3M). CG8483,
which has homology to a venom allergen, is expressed in a complex pattern
suggestive of expression in peripheral sense organ precursors
(Fig. 3W).
We determined the expression pattern for five of eight genes for which the sequence reveals no homology to known protein domains. CG15489 and CG15488 (Fig. 2I,K) are in a cluster of genes also including nub and pdm-2 that are expressed in similar domains and are adjacent in the genome. CG15001, consisting of only a single exon, is adjacent to another gene (CG15000), also discovered on the arrays, with a similar expression domain (Fig. 3H,L). BG:DS00797.2/CG9008 is expressed strongly in the wing pouch and also in the adepithelial cell layer (Fig. 3N). CG8780 is highly enriched on the arrays (31-fold, Table 2) and expressed specifically in the hinge and ventral pleura (Fig. 3D).
Regulation by wg signaling
The genes, CG17278, Cyp310a1 and CG8381 all show very
intense expression in the wing pouch but reduced expression at the DV margin
(Fig. 4A,D,G). Wg is expressed
at the DV margin forming a gradient that regulates the expression of target
genes in a concentration-dependent manner
(Strigini and Cohen, 2000). To
determine whether Wg signaling represses the expression of CG17278,
Cyp310a1 and CG8381, we ectopically expressed wg in the
dorsal and ventral wing-pouch regions (71B-gal4; UAS-wg), or
inhibited Wg function at the DV margin by expressing a dominant-negative form
of TCF (van de Wetering et al.,
1997
), a transcription factor required for Wg-signal transduction
(C96-GAL4; UAS-DN-dTCF). With higher levels of Wg activity
in the wing pouch, expression of all three genes was inhibited
(Fig. 4B,E,H). In contrast,
inhibition of Wg signaling at the DV margin allowed ectopic expression of
Cyp310a1 in all margin cells and increased the number of cells
expressing CG17278 and CG8381
(Fig. 4F,C,I). In the
presumptive margin, cells continue to express wg in the absence of Wg
activity, cell replication increases
(Phillips and Whittle, 1993
),
and ectopic expression of dmyc appears in margin cells
(Johnston et al., 1999
).
Therefore, ectopic expression of the genes studied here is caused by loss of
Wg-dependent repression rather than loss of the non-expressing cells from the
presumptive margin. This does not imply that Wg-dependent repression must be
direct. Without functional data on these potential target genes, their
relationship to wg and their role in wing patterning remain
unknown.
|
Concluding remarks
We have shown that microarray analysis of RNA profiles, followed by in situ
hybridization, can rapidly identify candidate genes that warrant investigation
for their role in wing disc development. Most genes identified here are not
represented by mutant alleles. This may reflect any of several situations. (1)
Some genes may be refractive to mutagenesis. This is unlikely to be the case
for chemical mutagens or ionizing radiation, but P elements show specificity
in insertion site (Liao et al.,
2000). As P elements are the mutagen of choice in most current
screens, for example, the Berkeley Drosophila Genome Project (BDGP) screen
(Spradling et al., 1999
), some
genes may not be susceptible. (2) Redundancy masks gross phenotypes. This may
be a factor for highly related genes such as Doc1 and
Doc2/CG5187, which are also expressed in very similar patterns
(Fig. 3R,X), and for members of
multi-gene families such as the cytochrome, Cyp310a1 and the serpin,
CG17278. (3) The genes have no crucial function. Despite having a
localized expression pattern, some genes may play a minor role or no role in
the cells in which they are expressed.
The challenge will be to decide among these possibilities in a new round of
genetic analysis that uses techniques such as RNA silencing or homologous
recombination to reduce function of specific genes
(Fortier and Belote, 2000;
Kennerdell and Carthew, 1998
;
Kennerdell and Carthew, 2000
;
Martinek and Young, 2000
;
Piccin et al., 2001
;
Rong and Golic, 2000
;
Rong et al., 2002
), and if
necessary simultaneously in several genes in a family, to determine if
phenotypic change occurs. Whole genome profiling is a powerful method to
identify genes that then become a high priority for such analysis.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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REFERENCES |
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