The Drosophila wing disc is divided along the proximaldistal
axis into regions giving rise to the body wall (proximal), wing hinge
(central) and wing blade (distal). DNA microarray analysis has been applied to
discover genes with potential roles in the development of these regions. A set of 94 transcripts, enriched two fold or greater, were identified in the body
wall and 56 enriched transcripts in the wing/hinge region. Transcripts
that are known to have highly restricted expression patterns, such as
pannier, twist and Bar-H1 (body wall) and knot,
nubbin and Distal-less (wing/hinge), show strong differential
expression on the arrays. In situ hybridization for 50 previously
uncharacterized genes similarly revealed that transcript enrichment identified
by the array analysis is consistent with the observed spatial expression.
There was a broad spectrum of patterns, in some cases suggesting that the
genes could be targets of known signaling pathways. Three of
these genes respond to wingless signaling. Genes
likely to play specific roles in tracheal and myoblast cell types were also discovered, since these cells are part of the body wall fragment. In summary, the identification of
genes with restricted expression patterns using whole genome profiling
suggests that many genes with potential roles in wing disc development remain
to be characterized (Butler, 2003).
To identify genes with expression patterns enriched in the presumptive
wing/hinge or body wall regions, wing imaginal discs were cut into two
fragments at the boundary between the body wall and the wing hinge. Folds associated with the hinge provide morphological features to allow precise cutting. RNA expression
profiles of these samples were determined using oligonucleotide microarrays
representing approximately 13,500 known and predicted genes in the
Drosophila genome (Genechip Drosophila Genome Array 1,
Affymetrix). Information for all genes is available at http://dev.biologists.org/supplemental/. Ninety-four transcripts show two-fold or greater enrichment in the body wall and 56 transcripts show two-fold or greater enrichment in the wing/hinge. Several of these
genes were also found to be more highly expressed in
wing discs than leg discs or eye-antennal discs, suggesting they may also have
appendage-specific roles (Butler, 2003).
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, are all known to be highly expressed in the presumptive body wall. 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. kn is expressed in the wing 3/4 intervein and hinge regions; nub is strongly expressed in the entire wing pouch and
Dll is expressed along the dorsal-ventral (DV) margin exclusively in
the wing pouch (Butler, 2003).
Other genes, known to have important roles in disc development, appear
lower down the rank order. vestigial (vg), a key gene for development
of the wing and hinge regions, 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. Transcripts with expression patterns restricted to the
posterior compartment, [engrailed (en), invected
(inv) and hedgehog (hh)], show
approximately two-fold enrichment in the wing/hinge sample. 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. 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. The m6 gene is an exception (enriched in the body wall sample) and is known to be
expressed only in the body wall region. In
contrast, genes that show ubiquitous expression such as Ras or tubulin show no
enrichment on the arrays (Butler, 2003).
Microarray analysis can therefore identify transcripts known to be
differentially expressed in the wing/hinge and body wall regions of the disc. Few expression patterns of the identified genes 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, in situ hybridizations were made for some of these uncharacterized genes (Butler, 2003).
Fifty transcripts that had strong enrichment
(mostly three-fold or greater) were examined. For the body wall-enriched transcripts, the larger set (only transcripts for which clones are available in the
Drosophila gene collections -- DGC1 and DGC2, Berkeley Drosophila
Genome Project) were examined. For the wing/hinge region, transcripts were examined with
three-fold or greater enrichment, systematically in rank order from the top,
and PCR probes were generated when clones were not available. 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 (Butler, 2003).
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. The adepithelial
layer extends from the proximal disc dorsally into the hinge region. The body wall
fragment includes cells of all three layers, so the arrays also identified
transcripts specific to muscle and tracheal cells (Butler, 2003).
pan and BarH1, which encode transcription factors, are
expressed in the body wall epidermis and are involved in bristle patterning. Both
transcripts were highly enriched on the arrays. Also highly enriched
was tailup (tup), 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. 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. 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, CG10126, CG3244 and Glucose dehydrogenase. Obp56a/CG11797 encodes an odorant-binding protein and interestingly three other odorant-binding proteins showed enrichment on the arrays: Obp99a, CG9358 and Obp56d/CG1128. Idgf4, encoding an imaginal disc growth factor, is
expressed in the peripodial membrane, primarily in dorsal cells. Presumably secretion
of Idgf4 could influence development of the columnar epithelium (Butler, 2003).
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: Mef2,
twist (twi) and heartless (htl).
Act57B is known to be regulated by Mef2 in the embryo, and Act57B is expressed in the myoblasts, suggesting this
relationship also exists in these adult muscle precursors. Mef2
expression is activated by twi and may
be inhibited by the transcriptional repressor, zinc finger homology 1
(zfh1). zfh1 is expressed in the myoblasts. stumps is
also enriched on the arrays and expressed in the myoblasts.
Together with htl, stumps has a role in the development of the
tracheal cells. 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. Both transcripts are enriched on the arrays and show similar
expression patterns in the adepithelial myoblasts and blood cells. Other genes
showing specific expression in the myoblasts are BM-40/SPARC, a
calcium-binding glycoprotein, which is expressed in the embryonic mesoderm, Elongation factor 1 alpha 100E (Ef1 alpha), CG8689, an
alpha-amylase, and two transcripts encoding predicted proteins with unknown function
CG11100 and CG15064 (Butler, 2003).
In the wing disc, cells of the larval and developing adult tracheal systems
require activity of genes in the FGF pathway.
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). 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. However, it is not clear why the arrays failed to detect btl expression because six genes were identified that are also expressed specifically in tracheal cells -- these are CG5397, an
O-acyltransferase, CG4386, a serine-type endopeptidase, CG2663, an
alpha-tocopherol transfer-like protein, and CG15353, CG6921 and CG9338 that have no known homologies. In particular, CG4386 is interesting since it is only expressed in the dorsal branch, and CG6921 is distinguished because it is very strongly expressed in the most proximal cells (Butler, 2003).
The wing/hinge fragment of the wing disc primarily contains cells of the
peripodial membrane and the columnar epithelium, with only a few
myoblasts that extend into the hinge region. Thus the genes detected by the
arrays as enriched in this disc fragment are expressed in cells of one of the
two epithelial layers (Butler, 2003).
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. pdm2, which is highly related to nub,
also shows wing-enriched expression on the arrays and is expressed in a
similar domain to nub. pdm2 apparently has no significant function in the
wing. The roles of the remaining seven predicted transcription factors are 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. defective proventriculous (dve), which
encodes a homeodomain protein, 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. odd
paired (opa), known for a role in embryonic segmentation, is
discretely expressed in cells of the presumptive mesopleura and dorsal hinge. No role for opa in wing disc development has been reported. Dorsocross1
(Doc1) and Doc2/CG5187 are T-Box related factors that are
expressed in what appears to be an identical domain in the wing disc. Both transcripts
also accumulate in body wall cells and this probably lowers their position in
the overall ranked list (Butler, 2003).
Eight transcripts encoding enzymes are enriched two-fold or greater in the
wing/hinge region. This group includes the most highly enriched transcript detected in the analysis, a kazal-type serpin gene CG17278 (68-fold). 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. Variable expression in anterior body wall cells is also
observed that is consistent with the array data that indicate
Cyp310al transcripts are also present in body wall RNA. Surprisingly, the
ß-galactosidase gene (CG3132) was found to be enriched in the
wing/hinge region. Weak expression was found 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. Thus the ß-gal transcript probably appears as wing/hinge enriched primarily because it is expressed in blood cells. The
expression patterns of two other enzymes were also determined: the metalloendopeptidase
Nep1/CG5894 and UDP-glucosyl transferase (Ugt86Di) (Butler, 2003).
The alpha-integrin, inflated, which has a role in cell adhesion,
is expressed in the ventral compartment and is
thus enriched on the wing/hinge arrays. A novel gene,
CG5758, is potentially involved in cell adhesion since it encodes a
predicted protein with ß-Ig-H3/Fas domains and its expression is
restricted to the dorsal hinge. 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. Despite intense expression in the wing pouch, CG8381 shows only modest enrichment on
the arrays, probably reflecting the fact that the gene is also expressed in several groups of cells in the body wall region (Butler, 2003).
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. wengen
/CG6531, which is a receptor of the TNFR family, is
expressed strongly in the wing pouch and weakly in the body wall. On the arrays, its
ligand, eiger, was undetectable in the wing/hinge region sample but
enriched in the body wall sample (Butler, 2003).
Two structural proteins, CG6469, a larval cuticle protein, and
CG14301, a chitin-binding protein, are the only genes 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. 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 (Butler, 2003).
In a group of genes with miscellaneous functions the expression of three genes was determined. anachronism (ana), a secreted glycoprotein, is expressed in five clusters of cells including one in the body wall region and in some individual neuroblasts. ana null mutants are viable and have no observable defects suggesting it is not required, or functions redundantly, in the wing.
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. CG8483,
which has homology to a venom allergen, is expressed in a complex pattern
suggestive of expression in peripheral sense organ precursors (Butler, 2003).
The expression pattern is described for five of eight genes for which the
sequence reveals no homology to known protein domains. CG15489 and
CG15488
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.
BG:DS00797.2/CG9008 is expressed strongly in the wing pouch and also
in the adepithelial cell layer. CG8780 is highly enriched on the arrays (31-fold), and expressed specifically in the hinge and ventral pleura (Butler, 2003).
The genes CG17278, Cyp310a1 and CG8381 all show very
intense expression in the wing pouch but reduced expression at the DV margin. Wg is expressed at the DV margin forming a gradient that regulates the expression of target
genes in a concentration-dependent manner. To
determine whether Wg signaling represses the expression of CG17278,
Cyp310a1 and CG8381, wg was ectopically expressed in the
dorsal and ventral wing-pouch regions (71B-gal4; UAS-wg), or Wg function
was inhibited at the DV margin by expressing a dominant-negative form
of TCF (Pangolin), 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. 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. In the
presumptive margin, cells continue to express wg in the absence of Wg
activity; cell replication increases,
and ectopic expression of dmyc appears in margin cells.
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 (Butler, 2003).