1 Department of Biochemistry and Biophysics, University of California, San
Francisco, CA 94143, USA
2 Department of Biology, University of Washington, Seattle, WA 98195, USA
Author for correspondence (e-mail:
tkornberg{at}biochem.ucsf.edu)
Accepted 2 June 2005
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SUMMARY |
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Key words: Transdetermination, Cellular plasticity, Imaginal disc, Polycomb group, Trithorax group, Expression profiling
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Introduction |
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Developmental plasticity has been observed and studied in many systems, but it has attracted increased interest recently because of the potential medical applications of human stem cells. In Drosophila, the phenotype of the classic homeotic mutants, which grow normal body parts in inappropriate locations, indicates that cells choose between developmental pathways and have the capacity to follow more than one. Changing the activity of these homeotic genes late in development results in similar mutant phenotypes, indicating that cells retain the capacity to change their determined state long after they adopt a commitment. Developmental plasticity in the context of losing or activating gene functions in individual cells has been extensively characterized in this system. The significance of transdetermination is founded in the likelihood that changes of determined state have an epigenetic basis, providing access to influences that establish or are needed to maintain determined states.
There are strong parallels between the processes of homeosis and
transdetermination in Drosophila and similar processes in mammals.
Homeotic transformations of vertebrates that are comparable to the homeotic
mutations in Drosophila have been described. One is Barrett's
esophagus, which results in the formation of intestinal tissue from the
esophageal epithelium (Jankowski et al.,
1999). Interestingly, ectopic activation of the Wnt signaling
pathway [orthologous to fly Wingless (Wg)] in the lungs of mouse embryos
results in the appearance of cells in the lung that have histological and
molecular characteristics of intestinal epithelial cells
(Okubo and Hogan, 2004
).
Hyperactivation of the Wnt signaling pathway also promotes cell fate switches
in epidermal and hair follicle cells
(Merrill et al., 2001
;
Niemann et al., 2002
) and in
the mammary gland and prostate (Bierie et
al., 2003
; Miyoshi et al.,
2002
).
When prothoracic (1st) leg discs are fragmented and cultivated in vivo,
cells in a proximodorsal region known as the `weak point' can switch fate and
transdetermine. These `weak point' cells give rise to cuticular wing
structures (Strub, 1977). The
leg-to-wing switch is regulated, in part, by the expression of the
vestigial (vg) gene, which encodes a transcriptional
activator that is a key regulator of wing development
(Kim et al., 1996
;
Williams et al., 1991
).
vg is not expressed during normal leg development, but it is
expressed during normal wing development and in `weak point' cells that
transdetermine from leg to wing (Johnston
and Schubiger, 1996
; Maves and
Schubiger, 1995
). Activation of vg gene expression marks
leg-to-wing transdetermination.
Sustained proliferation appears to be a prerequisite for fate change, and
conditions that stimulate growth increase the frequency and enlarge the area
of transdetermined tissue (Schubiger,
1973; Schweizer and
Bodenstein, 1975
; Tobler,
1966
). As noted above, transdetermination was discovered when
fragments of discs were allowed to grow for an extensive period of in vivo
culture. More recently, ways to express Wg ectopically have been used to
stimulate cell division and cell cycle changes in `weak point' cells
(Sustar and Schubiger, 2005
),
and have been shown to induce transdetermination very efficiently
(Maves and Schubiger, 1998
).
In the work reported here, we designed experiments to characterize the genes
involved in or responsible for transdetermination that was induced by ectopic
Wg. We focused on leg-to-wing transdetermination because it is well
characterized, it can be efficiently induced and it can be monitored by the
expression of a real-time GFP reporter. These attributes make it possible to
isolate transdetermining cells as a group distinct from dorsal leg cells,
which regenerate, and ventral leg cells in the same disc, which do not
regenerate; and, in this work, to directly define their expression profiles.
Our analysis identified unique expression properties for each of these cell
populations. It also identified a number of genes whose change in expression
levels may be significant to understanding transdetermination and the factors
that influence developmental plasticity. One is lamina ancestor
(lama), whose expression correlates with undifferentiated cells and
we show controls the area of transdetermination. Another has sequence
similarity to the mammalian augmenter of liver regeneration (Alr;
Gfer Mouse Genome Informatics), which controls regenerative
capacity in the liver and is upregulated in mammalian stem cells. We also
found that fifteen regulators of chromatin structure [e.g. members of the
Polycomb group (PcG) and trithorax group (trxG)] are differentially regulated
in transdetermining cells and that mutants in seven of these genes have
significant effects on transdetermination. These studies identify two types of
functions that transdetermination requires functions that promote an
undifferentiated cell state and functions that re-set chromatin structure.
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Materials and methods |
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Microarray data is available at http://www.ncbi.nlm.nih.gov/projects/geo/index.cgi and can be accessed using the series ID number GSE2886.
Induction of transdetermination and culture of discs
Ectopic Wg expression was induced by heat shock either at 60 hours or 72
hours after egg deposition, and leg discs were isolated 3 days later. Wg
overexpression delays metamorphosis by 1 day, so these larvae had not yet
pupariated. For the regeneration experiments, the `3/4 fragment' of 2nd leg
discs were isolated and cultured in the abdomen of adult female flies for 3-5
days.
Sample isolation and RNA amplification
Leg discs were dissected in PBS and either used as whole discs or were cut
with tungsten needles to isolate the three cell populations: GFP-expressing
TD, DWg and VWg, as indicated in
Fig. 2A. Linear RNA
amplification was performed essentially as described
(Klebes et al., 2002). In
brief, RNA was extracted from single imaginal discs or from pooled fragments
from four to eight discs using the Mini RNA isolation kit (Zymo Research).
Total RNA was used for a first round of reverse transcription and in vitro
transcription using T7 RNA polymerase. Subsequently, the amplified RNA product
was subjected to a 2nd round of reverse transcription and in vitro
transcription yielding 10,000-fold amplification or more.
Production of microarrays, hybridization and composition of reference sample
Amplified RNA was indirectly labeled by a reverse transcription reaction in
the presence of amino-allyl-modified dUTP
(Klebes et al., 2002)
(www.microarrays.org).
Fluorescent dyes (Cy3 and Cy5, Amersham) were coupled to the modified
nucleotides. Data was collected using a GenePix scanner 4000A (Axon
Instruments). Microarrays were produced as described
(Xu et al., 2003
). In brief,
14,151 specific primer pairs (Incyte Genomics) were used for PCR amplification
of 100-600 bp long fragments of annotated open reading frames. The common
reference sample to which we compared control leg imaginal discs, Wg-induced
whole leg imaginal discs and all experimental samples of the regeneration
group was generated by pooling amplified RNA of male and female 1st, 2nd and
3rd leg imaginal discs in equal proportions. For about half of the
experiments, dyes were reversed to avoid bias (see details in Table S1).
Data analysis
Scanned images were processed using GenePix software (Axon Instruments).
Signal intensities were further processed as expression ratios
(log2 transformed). Global median normalization was performed on
NOMAD
(www.microarrays.org).
Genes with expression levels smaller than 350 as the sum of medians were not
included. Cluster analysis was performed with Cluster software and visualized
with Treeview (Eisen et al.,
1998) with the filtering settings detailed in
Fig. 3. The median expression
ratios of replicate experiments was calculated in Excel (Microsoft). To
determine the median expression, replicate experiments were filtered
threefold: (1) data for a given gene had to be present in at least 60% of all
replicate experiments; (2) the P-value (heteroscedastic, two-tailed
student's t-test) had to be less than 0.05 (95% confidence level);
and (3) the median expression level had to be greater than 1.85-fold (0.8,
log2). To correct for multiple testing the comparisons of wild-type
wing and leg discs and the Wg-induced TD, DWg, and VWg
cells were subjected to the significance analysis of microarrays software
package (Tusher et al., 2001
)
and all genes from the median-based lists were determined to be
significant.
Analysis of PcG and trxG expression levels was performed with a two-sided
t-test using the limma package in the statistical software R
(Ihaka and Gentleman, 1996). A
`moderated' t-statistic was calculated to account for small sample sizes and
differences in variability of expression values between genes
(Smyth, 2004
). To correct for
multiple testing, the P values from the t-test were adjusted
by controlling the false discovery rate
(Benjamini et al., 2001
).
Ratios within a 95% confidence level and a median ratio greater 0.25
(log2) were considered significant. Fifteen out of 32 (47%) of the
genes satisfy the two conditions. Among all of the genes in these experiments
(n=11.952), 14.4% satisfy these conditions (binomial test,
P-value=1.140x105).
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Results |
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In order to distill the genes involved in transdetermination from the expression profiles of more than 14,000 genes of TD cells, we generated nine different categories of samples for control and comparative purposes (see Table S1): (1) wild-type wing (W); (2) leg (L) discs of 3rd instar larvae; (3-6) dorsal and ventral cells from both wild-type (DWT and VWT) and Wg-expressing (DWg and VWg) discs; (7) intact Wg-induced 1st leg discs; (8) micro-dissected TD cells from these discs (TD); and (9) fragments of 2nd leg discs that were cultivated to obtain a population of regenerating cells. Last, we prepared a `reference sample' of wild-type leg discs (see Materials and methods). DNA arrays were hybridized to pairs of probes that had been generated from the same larva or, when this was impractical, to the common reference sample.
Expression profiles of wild-type wing and leg imaginal discs
Probes prepared from wing and leg discs were hybridized together to arrays
and the median expression ratios were compared. Using a stringent filter
setting (see Materials and methods), 67 wing-specific and 62 leg-specific
transcripts were identified (Table
1, see Table S2 in the supplementary material). Genes in the wing
cluster previously shown to be expressed most abundantly in wing discs
included collier, apterous and vg
(Diaz-Benjumea and Cohen,
1993; Vervoort et al.,
1999
; Williams et al.,
1991
). In situ hybridization with probes for transcripts from five
genes in the wing list and four genes in the leg list detected expression in
leg and wings discs that was consistent with the array experiments
(Fig. 1). Interestingly, a
significant proportion of the genes in the wing and leg lists encode
transcription factors. Among the 20 genes with the highest levels of
differential expression, seven (35%) in the wing list and nine (45%) in the
leg list encode transcription factors. To put this result in context,
5%
(715/14,113) of the genes in the annotated database are predicted to encode
transcription factors (Adams et al.,
2000
). If the wing- and leg-specific transcription factors have
multiple targets, most of their targets apparently are not differentially
expressed with comparable specificity.
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Expression profiles of regenerating cells
Expression profiles characteristic of regenerating cells were obtained by
analyzing disc fragments that had been cultured to promote proliferation. To
minimize possible contamination with transdetermined cells, we did not use 1st
leg discs, but instead examined the `3/4 fragment' of 2nd leg discs. This
fragment regenerates missing parts, and in contrast to 1st leg discs, does not
transdetermine (G.S., unpublished). The disc fragments were collected after
3-5 days of culture, a period that corresponds to the stage when
transdetermined cells express the vgBE-GFP marker and the stage when we
analyzed TD cells of 1st leg discs. Transcripts enriched in regenerating disc
fragments encoded proteins involved in protein synthesis, cytoskeletal
organization and energy metabolism (see Table S4 in the supplementary
material), an array of functions that is consistent with the increased mitotic
activity of proliferating cells. Seven genes out of the 130 (5.4%) that were
upregulated at least 1.8-fold in the regenerating cells encode transcription
factors.
We call attention to two genes in the regeneration cluster.
regucalcin is expressed in hemocytes associated with wing but not in
leg discs (Fig. 1E). Its
expression has been detected at sites of wound healing
(Vierstraete et al., 2004);
its elevated expression in regenerating cells (sixfold) may suggest that
hemocytes are recruited to sites of regeneration as well. headcase
(hdc) was also upregulated in the regenerating fragments (7.3-fold).
hdc is expressed in all imaginal discs during normal development and
has been characterized as a negative regulator of terminal differentiation
(Weaver and White, 1995
).
Suppression of terminal differentiation may be an essential step during
regeneration. As described in the next section, analysis of the lama
gene provides support for this suggestion.
Expression profiles of transdetermining cells
We used two methods to analyze the transcripts in TD cells. The first
entailed direct comparisons of the expression ratios. We compared GFP-positive
TD cells with DWg and VWg cells that we dissected from
the same 1st leg discs. One-hundred and forty-three `TD' genes whose
expression is enriched in TD cells were identified
(Table 1; see Table S5 in the
supplementary material). Of these genes, 19 are also upregulated in either
dorsal cells (DWg + Dwt) or regenerating cells. Fifteen
genes are also upregulated in wing cells (see Table S5 in the supplementary
material). The 109 genes in the TD set that are not characteristic of either
dorsal, regenerating or wing disc cells are implicated in
transdetermination.
We also analyzed the expression profiles of TD cells by hierarchical
clustering (Eisen et al.,
1998), a method that groups genes with expression levels that
change in similar ways. Clustering analysis confirmed that TD cells have a
distinct expression profile (see Fig.
3; see Tables S7 and S8 in the supplementary material). The
threshold settings for the two methods of analysis were different, but the
majority of TD genes (66%) were also grouped together by the clustering
routine. Among the many genes identified by this analysis, we focus this
description on the following.
CG14059
CG14059 is the gene in the transdetermination list whose expression
differed most dramatically (26.7-fold as a median of eight
TD-to-DWg and TD-to-VWg comparisons;
Table 1). In situ hybridization
confirmed these differences, detecting CG14059 RNA only in the transdetermined
region of Wg-expressing leg discs (Fig.
2B), but not in either wild-type leg or wing discs (not shown).
CG14059 is predicted to encode a novel protein that shares 77% sequence
identity with a conserved ortholog in D. pseudoobscura.
unpaired (upd; outstretched FlyBase; CG5993)
upd expression is significantly upregulated in TD cells
(12.3-fold; Table 1). It
encodes a ligand that activates the JAK/STAT signaling cascade
(Harrison et al., 1998). Two
aspects are consistent with a role for upd in the plasticity of the
TD cells. First, Upd regulation of the JAK/STAT pathway is essential for
suppressing differentiation and for promoting self-renewal of stem cells in
the Drosophila testis (reviewed by
Hombria and Brown, 2002
).
Second, upd interacts genetically with hdc
(Bach et al., 2003
). Although
we have not tested whether upd mutants affect transdetermination, the
enhanced expression of upd suggests that the JAK/STAT pathway might
play an important role.
apterous (ap; CG8376)
Ap is a LIM-homeodomain-containing protein whose function is essential to
wing development (Cohen et al.,
1992). It is expressed in normal leg discs in the presumptive
cells of the 4th tarsal segment (Pueyo et
al., 2000
), and is expressed at higher levels in wing discs
(10.8-fold; Table 1), where it
functions as a selector gene in dorsal cells
(Diaz-Benjumea and Cohen,
1993
). ap expression was marginally enhanced in TD cells,
and in our cluster analysis, ap segregated with the genes that were
upregulated in the wing disc. Anti-Ap antibody did not detect protein in
Wg-induced discs, but robust staining was observed in disc fragments that had
been cultivated in vivo (Fig.
2E). Antibody staining was detected in the same region as vgBE-GFP
expression, indicating that transdetermined cells express Ap. Although
transdetermined cells induced by Wg expression or fragmentation share many
genetic and cytological features (Johnston
and Schubiger, 1996
; Sustar
and Schubiger, 2005
), the differences in ap expression
indicate that they are not identical.
lim1 (CG11354)
Lim1 is another LIM-homeodomain containing protein that is expressed in
normal leg discs in a region that is distal to the ap expression
domain. Lim1 functions in concert with Ap to specify distal leg development
(Pueyo et al., 2000). Our
array analysis showed that lim1 expression is leg-specific (13-fold;
Table 1) and is up-regulated in
DWg cells (5.1-fold; not included in Table S3 because of the
insufficient number of data points). DWg cells are adjacent to the
TD cells, and expression of lim1 in Wg-expressing discs mimics the
adjacent expression domains of ap and lim1 in wild-type leg,
and suggests a functional interaction between the TD cells and the adjacent
leg cells (Fig. 2F,G).
CG4914
CG4914 is predicted to encode a protein that may have serine protease,
DNA-binding and/or transcription factor activities. Array hybridization showed
a 4.6-fold enrichment in transcript levels in wing-to-leg comparisons
(Table 1), and in situ
hybridization corroborated the higher level of expression in normal wing discs
(Fig. 1D). Its designation as a
marker of cells that switch to wing fate was validated by showing that its
expression in wg-expressing leg discs was most abundant in the
transdetermining region (Fig.
2C).
CG12534
Expression of CG12534 was enriched 2.3-fold in TD cells (see Table S5 in
the supplementary material). Although expression of CG12534 was also enriched
3.8-fold in three experiments analyzing regenerating 2nd leg discs, it did not
pass the filter settings for inclusion in the regeneration list (no
hybridization signal was obtained in one of four replicate experiments).
CG12534 has sequence homology with the mouse gene augmenter of liver
regeneration (Alr). Both CG12534 and Alr encode conserved
ERV1 domains (Lisowsky et al.,
1995). ALR has been implicated as a growth factor that contributes
to the regenerative capacity of mammalian liver
(Hagiya et al., 1994
) and
pancreas (Adams et al., 1998
),
and expression of the Alr gene has been found to be common to mouse
embryonic stem cells, neural stem cells and hematopoietic stem cells
(Ramalho-Santos et al., 2002
).
CG12534 is the only gene we found that TD cells have in common with both
regenerating cells and the three types of mouse stem cells. We examined
CG12534 expression by in situ hybridization. In wild-type wing and leg discs,
no expression was detected (not shown). However, in leg discs that ectopically
express Wg, we confirmed that CG12534 is expressed most abundantly in
vgBE-GFP-expressing TD cells (Fig.
2D). We suggest that CG12534 may encode an evolutionarily
conserved function in the regenerative process. Functional tests with
knock-down mutants are in progress.
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To investigate whether lama plays a role in transdetermination, we
first examined lama expression during normal larval development. In
situ hybridization revealed that lama is not expressed in late 3rd
instar discs, but is expressed uniformly by early 3rd instar discs, in the
imaginal ring of the proventriculus and in the salivary gland
(Fig. 4). Functional tests were
performed in the viable, null mutant lama410
(Perez and Steller, 1996) by
monitoring vgBE-lacZ expression in Wg-expressing mutant discs as an
indicator of transdetermination. Compared with controls, the frequency of
transdetermination was unchanged. However, the relative fraction of the leg
disc that expressed vg decreased from 5% to 2%
(Fig. 5). Expression of
lama in early, but not late discs suggests that the role of
lama in normal development may be to suppress pathways that promote
differentiation, and the significant decrease in transdetermined region of
lama mutant discs suggests that it may preserve the pluripotency of
disc cells in Wg-expressing discs.
Notch
Levels of Notch expression did not change significantly. However,
the cluster analysis identified four genes that encode proteins with roles in
Notch signaling: Enhancer of split, E(spl) region transcript m7, E(spl)
region transcript m2 and Serrate, whose expression decreased;
and two genes, Notchless and bancal, with increased levels
in TD cells. As the vgBE enhancer includes a binding site for Suppressor of
Hairy [Su(H)], a transcription factor that acts downstream of Notch and is
required for vg expression (Couso
et al., 1995), Notch signaling may contribute to activation of the
vgBE enhancer in TD cells. There may be additional inputs from Notch
signaling, as wing fate is among the many cell fate decisions that Notch
signaling regulates (Kurata et al.,
2000
).
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Members of the PcG and trxG are known to act as heteromeric complexes by
binding to cellular memory modules (CMMs). Our functional tests demonstrate
that mutant alleles for members of both groups have the same functional
consequence (they increase transdetermination frequency). Our findings are
consistent with recent observations that the traditional view of PcG members
as repressors and trxG factors as activators might be an oversimplification,
and that a more complex interplay of a varying composition of PcG and trxG
proteins takes place at individual CMMs (reviewed by
Lund and van Lohuizen, 2004).
Furthermore the opposing effects of Pc and Su(z)2 functions are consistent
with the proposal that Su(z)2 is one of a subset of PcG genes that is required
to activate as well as to suppress gene expression
(Gildea et al., 2000
). In
addition to measuring the frequency of transdetermination, we also analyzed
the relative area of vg expression in the various PcG and trxG
heterozyogous mutant discs. As shown in
Fig. 5B (see also Table S11 in
the supplementary material), the relative area decreased in E(Pc),
brm and osa mutant discs, despite the increased frequency of
transdetermination in these mutants. We do not have evidence to explain these
contrasting effects, but the roles of these seven PcG and trxG genes in
transdetermination that these results identify support the proposition that
the transcriptional state of determined cells is implemented through the
controls imposed by the regulators of chromatin structure.
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We also analyzed the upstream regions of the genes whose transcripts were enriched in D. melanogaster TD cells, using a computational supervised search for vgBE-like motifs. Seventeen genes contain one or more clusters of Sd-, Pan- and Su(H)-binding site sequences and 45 had putative binding sites for at least one of these transcription factors (see Table S10 in the supplementary material). Among the TD genes with binding sites are vg, CG14059 and CG12534. Verification that these sequences bind Sd, Pan and Su(H) is necessary, but the presence of these sequences is consistent with the expectation that many of the TD genes are directly regulated by the Vg, Wg and Notch pathways.
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Discussion |
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The general issue these studies address is the molecular basis for changes in cell fate, a subject we tackled by analyzing transcriptional profiles to ask which genes instigate and elaborate such changes. The microarray experiments we performed yielded relative quantitative data for as many as 75% of the predicted transcription units in the Drosophila genome, and we showed using in situ hybridization probes for a small number of genes that the predictive value of the data is excellent. Genes identified in the arrays to be expressed in a wing-specific or leg-specific manner, or to be differentially regulated in TD cells had expression patterns consistent with the array results. Importantly, tests of several genes implicated by differential expression in TD cells indicated that these genes function to either promote or inhibit transdetermination. In part, the value of these studies is the demonstration that the methods we used yield high quality data from small amounts of tissue that can be isolated by hand with little effort, making it possible to carry out replicate experiments for many conditions and samples. In work that is yet unpublished, we used these same methods to analyze other developmental systems in Drosophila for example, haltere discs transformed by various combinations of bithorax mutants, different larval tracheal metameres, various parts of the male reproductive apparatus, and anterior and posterior compartments of the wing disc (A.K. and T.B.K., unpublished). We suggest that these methods will be useful for studies of developmental programs in many other contexts as well. The method of analysis we used is not comprehensive, as the arrays do not query all the predicted protein coding sequences and the amplification technique we developed does not yield a complete representation of full-length transcripts. However, as we show in this work, the method constitutes a robust and high value screen that can be queried in various ways to identify candidate genes. The data we obtained is informative both about the general landscape of the transcriptional profiles and about individual genes.
Overlap between the transcriptional profiles in the wing and
transdetermination lists (15 genes) and with genes in subcluster IV
(Fig. 3) is extensive. The
overlap is sufficient to indicate that the TD leg disc cells have changed to a
wing-like program of development, but interestingly, not all wing-specific
genes were activated in the TD cells. The reasons could be related to the
incomplete inventory of wing structures produced (only ventral wing; G.S.,
unpublished) or to the altered state of the TD cells. During normal
development, vg expression is activated in the embryo and continues
through the 3rd instar. Although the regulatory sequences responsible for
activation in the embryo have not been identified, in 2nd instar wing discs,
vg expression is dependent upon the vgBE enhancer, and in 3rd instar
wing discs expression is dependent upon the vgQE enhancer
(Kim et al., 1996;
Klein and Arias, 1999
).
Expression of vg in TD cells depends on activation by the vgBE
enhancer (Maves and Schubiger,
1998
), indicating that cells that respond to Wg-induction do not
revert to an embryonic state. Recent studies of the cell cycle characteristics
of TD cells support this conclusion
(Sustar and Schubiger, 2005
),
but the role of the vgBE enhancer in TD cells and the incomplete inventory of
`wing-specific genes' in their expression profile probably indicates as well
that at the time that we analyzed the TD cells, they were not equivalent to
the cells of late 3rd instar wing discs.
Investigations into the molecular basis of transdetermination have led to a
model in which inputs from the Wg, Dpp and Hh signaling pathways alter the
chromatin state of key selector genes to activate the transdetermination
pathway (Maves and Schubiger,
2003). Our analyses were limited to a period 2-3 days after the
cells switched fate, because several cell doublings were necessary to produce
sufficient numbers of marked TD cells. As a consequence, these studies did not
analyze the initial stages. Despite this technical limitation, this study
identified several genes that are interesting novel markers of
transdetermination (e.g. ap, CG12534, CG14059 and CG4914), as well as
several genes that function in the transdetermination process (e.g.
lama and the PcG genes). The results from our
transcriptional profiling add significant detail to the general model proposed
by Maves and Schubiger (Maves and
Schubiger, 2003
).
First, we report that ectopic wg expression results in
statistically significant changes in the expression of 15 PcG and trxG genes.
Moreover, although the magnitudes of these changes were very small for most of
these genes, functional assays with seven of these genes revealed remarkably
large effects on the metrics we used to monitor transdetermination the
fraction of discs with TD cells, the proportion of disc epithelium that TD
cells represent, and the fraction of adult legs with wing cuticle. These
effects strongly implicate PcG and trxG genes in the process of
transdetermination and suggest that the changes in determined states
manifested by transdetermination are either driven by or are enabled by
changes in chromatin structure. This conclusion is consistent with the
demonstrated roles of PcG and trxG genes in the self-renewing capacity of
mouse hematopoietic stem cells (reviewed by
Valk-Lingbeek et al., 2004),
in Wg signaling and in the maintenance of determined states
(Barker et al., 2001
;
Collins and Treisman, 2000
;
Petruk et al., 2001
). Our
results now show that the PcG and trxG functions are also crucial to
pluripotency in imaginal disc cells, namely that pluripotency by `weak point'
cells is dependent upon precisely regulated levels of PcG and trxG proteins,
and is exquisitely sensitive to reductions in gene dose.
Our data do not suggest how the PcG and trxG genes affect
transdetermination, but several possible mechanisms deserve consideration. The
recent study of Sustar and Schubiger
(Sustar and Schubiger, 2005)
reported that transdetermination correlates with an extension of the S phase
of the cell cycle. Several proteins involved in cell cycle regulation
physically associate with PcG and trxG proteins
(Brumby et al., 2002
;
Trimarchi et al., 2001
), and
Brahma, one of the proteins that affects the metrics of transdetermination we
measured, has been shown to dissociate from chromatin in late S-phase and to
reassociate in G1. It is possible that changes in the S-phase of TD cells are
a consequence of changes in PcG/trxG protein composition.
Another generic explanation is that transdetermination is dependent or
sensitive to expression of specific targets of PcG and trxG
genes. Among the 167 Pc/Trx response elements (PRE/TREs) predicted to exist in
the Drosophila genome (Ringrose
et al., 2003), one is in direct proximity to the vg gene.
It is possible that upregulation of vg in TD cells is mediated
through this element. Another factor may be the contribution of targets of Wg
signaling, as Collins and Treisman reported that targets of Wg signaling are
upregulated in osa and brm mutants
(Collins and Treisman, 2000
).
These are among a number of likely possible targets, and identifying the sites
at which the PcG and trxG proteins function will be necessary if we are to
understand how transdetermination is regulated. Importantly, understanding the
roles of such targets and establishing whether these roles are direct will be
essential to rationalize how expression levels of individual PcG and
trxG genes correlate with the effects of PcG and
trxG mutants on transdetermination.
Second, the requirement for lama suggests that proliferation of TD
cells involves functions that suppress differentiation. lama
expression has been correlated with neural and glial progenitors prior to, but
not after, differentiation (Perez and
Steller, 1996), and we observed that lama is expressed in
imaginal progenitor cells and in early but not late 3rd instar discs
(Fig. 4). We found that
lama expression is re-activated in leg cells that transdetermine. The
upregulation of unpaired in TD cells may be relevant in this context,
as the JAK/STAT pathway functions to suppress differentiation and to promote
self-renewal of stem cells in the Drosophila testis (reviewed by
Hombria and Brown, 2002
). We
suggest that it has a similar role in TD cells.
Third, a role for Notch is implied by the expression profiles of several Notch pathway genes. Notch may contribute directly to transdetermination through the activation of the vgBE enhancer [which has a binding site for Su(H)] and of similarly configured sequences that we found to be present in the regulatory regions of 45 other TD genes (see Table S10 in the supplementary material). It will be important to test whether Notch signaling is required to activate these co-expressed genes, and if it is, to learn what cell-cell interactions and `community effects' regulate activation of the Notch pathway in TD cells.
Fourth, the upregulation in TD cells of many genes involved in growth and
division, and the identification of a DRE sites in the regulatory region of
many of these genes supports the observation that TD cells become
re-programmed after passing through a novel proliferative state
(Sustar and Schubiger, 2005),
and suggests that this change is in part implemented through DRE-dependent
regulation.
Two final comments: we are intrigued by the interesting correlation between
transdetermination induced by Wg mis-expression and the role of Wg/Wnt
signaling for stem cells. Wg/Wnt signaling functions as a mitogen and
maintains both somatic and germline stem cells in the Drosophila
ovary (Gonzalez-Reyes, 2003),
and mammalian hematopoetic stem cells
(Reya et al., 2003
). Although
the `weak point' cells in the Drosophila leg disc might lack the
self-renewing capacity that characterizes stem cells, they respond to Wg
mis-expression by manifesting a latent potential for growth and
transdetermination. It seems likely that many of the genes involved in
regulating stem cells and in leading to disease states when the relevant
regulatory networks lose their effectiveness are conserved.
We are also intrigued by the prevalence of transcription factors among the genes whose relative expression levels differed most in our tissue comparisons. It is commonly assumed that transcription factors function catalytically and that they greatly amplify the production of their targets, so the expectation was that the targets of tissue-specific transcription factors would have the highest degree of tissue-specific expression. In our studies, tissue-specific expression of 15 transcription factors among the 40 top-ranking genes in the wing and leg data sets (38%) is consistent with the large number of differentially expressed genes in these tissues, but these rankings suggest that the targets of these transcription factors are expressed at lower relative levels than the transcription factors that regulate their expression. One possible explanation is that the targets are expressed in both wing and leg disc cells, but the transcription factors that regulate them are not. This would imply that the importance of position-specific regulation lies with the regulator, not the level of expression of the target. Another possibility is that these transcription factors do not act catalytically to amplify the levels of their targets, or do so very inefficiently and require a high concentration of transcription factor to regulate the production of a small number of transcripts. Further analysis will be required to distinguish between these or other explanations, but we note that the prevalence of transcription factors in such data sets is neither unique to wing-leg comparisons (A.K. and T.B.K., unpublished) nor universal.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/16/3753/DC1
* Present address: Institut für Biologie, Genetik, Freie
Universität Berlin, 14195 Berlin, Germany
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, G. A., Maestri, M., Squiers, E. C., Alfrey, E. J., Starzl, T. E. and Dafoe, D. C. (1998). Augmenter of liver regeneration enhances the success rate of fetal pancreas transplantation in rodents. Transplantation 65, 32-36.[CrossRef][Medline]
Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A.,
Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A.,
Galle, R. F. et al. (2000). The genome sequence of
Drosophila melanogaster. Science
287,2185
-2195.
Adler, P. N., Charlton, J. and Brunk, B. (1989). Genetic interactions of the suppressor 2 of zeste region genes. Dev. Genet. 10,249 -260.[CrossRef][Medline]
Bach, E. A., Vincent, S., Zeidler, M. P. and Perrimon, N.
(2003). A sensitized genetic screen to identify novel regulators
and components of the Drosophila janus kinase/signal transducer and activator
of transcription pathway. Genetics
165,1149
-1166.
Baker, N. E. (1988). Transcription of the segment-polarity gene wingless in the imaginal discs of Drosophila, and the phenotype of a pupal-lethal wg mutation. Development 102,489 -497.[Abstract]
Barker, N., Hurlstone, A., Musisi, H., Miles, A., Bienz, M. and
Clevers, H. (2001). The chromatin remodelling factor Brg-1
interacts with beta-catenin to promote target gene activation. EMBO
J. 20,4935
-4943.
Benjamini, Y., Drai, D., Elmer, G., Kafkafi, N. and Golani, I. (2001). Controlling the false discovery rate in behavior genetics research. Behav. Brain Res. 125,279 -284.[CrossRef][Medline]
Berman, B. P., Nibu, Y., Pfeiffer, B. D., Tomancak, P.,
Celniker, S. E., Levine, M., Rubin, G. M. and Eisen, M. B.
(2002). Exploiting transcription factor binding site clustering
to identify cis-regulatory modules involved in pattern formation in the
Drosophila genome. Proc. Natl. Acad. Sci. USA
99,757
-762.
Bierie, B., Nozawa, M., Renou, J. P., Shillingford, J. M., Morgan, F., Oka, T., Taketo, M. M., Cardiff, R. D., Miyoshi, K., Wagner, K. U. et al. (2003). Activation of beta-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation. Oncogene 22,3875 -3887.[CrossRef][Medline]
Bornemann, D., Miller, E. and Simon, J. (1998).
Expression and properties of wild-type and mutant forms of the Drosophila sex
comb on midleg (SCM) repressor protein. Genetics
150,675
-686.
Brumby, A. M., Zraly, C. B., Horsfield, J. A., Secombe, J.,
Saint, R., Dingwall, A. K. and Richardson, H. (2002).
Drosophila cyclin E interacts with components of the Brahma complex.
EMBO J. 21,3377
-3389.
Bussemaker, H. J., Li, H. and Siggia, E. D. (2001). Regulatory element detection using correlation with expression. Nat. Genet. 27,167 -171.[CrossRef][Medline]
Cohen, B., McGuffin, M. E., Pfeifle, C., Segal, D. and Cohen, S. M. (1992). apterous, a gene required for imaginal disc development in Drosophila encodes a member of the LIM family of developmental regulatory proteins. Genes Dev. 6, 715-729.[Abstract]
Collins, R. T. and Treisman, J. E. (2000).
Osa-containing Brahma chromatin remodeling complexes are required for the
repression of wingless target genes. Genes Dev.
14,3140
-3152.
Corpet, F. (1988). Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16,10881 -10890.[Abstract]
Couso, J. P., Knust, E. and Martinez Arias, A. (1995). Serrate and wingless cooperate to induce vestigial gene expression and wing formation in Drosophila. Curr. Biol. 5,1437 -1448.[CrossRef][Medline]
Diaz-Benjumea, F. J. and Cohen, S. M. (1993). Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75,741 -752.[CrossRef][Medline]
Eisen, M. B., Spellman, P. T., Brown, P. O. and Botstein, D.
(1998). Cluster analysis and display of genome-wide expression
patterns. Proc. Natl. Acad. Sci. USA
95,14863
-14868.
Frith, M. C., Hansen, U. and Weng, Z. (2001). Detection of cis-element clusters in higher eukaryotic DNA. Bioinformatics 10,878 -889.[CrossRef]
Gildea, J. J., Lopez, R. and Shearn, A. (2000).
A screen for new trithorax group genes identified little imaginal discs, the
Drosophila melanogaster homologue of human retinoblastoma binding protein 2.
Genetics 156,645
-663.
Gindhart, J. G., Jr and Kaufman, T. C. (1995).
Identification of Polycomb and trithorax group responsive elements in the
regulatory region of the Drosophila homeotic gene Sex combs reduced.
Genetics 139,797
-814.
Gonzalez-Reyes, A. (2003). Stem cells, niches
and cadherins: a view from Drosophila. J. Cell Sci.
116,949
-954.
Hadorn, E. (1963). Differenzierungsleistungen wiederholt fragmentierter Teilstücke männlicher Genitalscheiben von Drosophila melanogaster nach Dauerkultur in vivo. Dev. Biol. 7,617 -629.[CrossRef]
Hadorn, E. (1965). Problems of determination and transdetermination. Brookhaven Symp. Biol. 18,148 -161.
Hagiya, M., Francavilla, A., Polimeno, L., Ihara, I., Sakai, H.,
Seki, T., Shimonishi, M., Porter, K. A. and Starzl, T. E.
(1994). Cloning and sequence analysis of the rat augmenter of
liver regeneration (ALR) gene: expression of biologically active recombinant
ALR and demonstration of tissue distribution. Proc. Natl. Acad.
Sci. USA 91,8142
-8146.
Halder, G., Polaczyk, P., Kraus, M. E., Hudson, A., Kim, J.,
Laughon, A. and Carroll, S. (1998). The Vestigial and
Scalloped proteins act together to directly regulate wing-specific gene
expression in Drosophila. Genes Dev.
12,3900
-3909.
Harrison, D. A., McCoon, P. E., Binari, R., Gilman, M. and
Perrimon, N. (1998). Drosophila unpaired encodes a secreted
protein that activates the JAK signaling pathway. Genes
Dev. 12,3252
-3263.
Hirose, F., Yamaguchi, M., Kuroda, K., Omori, A., Hachiya, T.,
Ikeda, M., Nishimoto, Y. and Matsukage, A. (1996). Isolation
and characterization of cDNA for DREF, a promoter-activating factor for
Drosophila DNA replication-related genes. J. Biol.
Chem. 271,3930
-3937.
Hombria, J. C. and Brown, S. (2002). The fertile field of Drosophila Jak/STAT signalling. Curr. Biol. 12,R569 -R575.[CrossRef][Medline]
Ihaka, R. and Gentleman, R. R. (1996). A language for data analysis and graphics. J. Comp. Graphic. Stat. 5,2999 -2344.
Jankowski, J. A., Wright, N. A., Meltzer, S. J.,
Triadafilopoulos, G., Geboes, K., Casson, A. G., Kerr, D. and Young, L. S.
(1999). Molecular evolution of the
metaplasia-dysplasia-adenocarcinoma sequence in the esophagus. Am.
J. Pathol. 154,965
-973.
Janson, K., Cohen, E. D. and Wilder, E. L. (2001). Expression of DWnt6, DWnt10, and DFz4 during Drosophila development. Mech. Dev. 103,117 -120.[CrossRef][Medline]
Johnston, L. A. and Schubiger, G. (1996).
Ectopic expression of wingless in imaginal discs interferes with
decapentaplegic expression and alters cell determination.
Development 122,3519
-3529.
Jones, R. S. and Gelbart, W. M. (1990). Genetic
analysis of the enhancer of zeste locus and its role in gene regulation in
Drosophila melanogaster. Genetics
126,185
-199.
Kim, J., Sebring, A., Esch, J. J., Kraus, M. E., Vorwerk, K., Magee, J. and Carroll, S. B. (1996). Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382,133 -138.[CrossRef][Medline]
Klebes, A., Biehs, B., Cifuentes, F. and Kornberg, T. B. (2002). Expression profiling of Drosophila imaginal discs. Genome Biol. 3,R0038 .
Klein, T. and Arias, A. M. (1999). The
vestigial gene product provides a molecular context for the interpretation of
signals during the development of the wing in Drosophila.
Development 126,913
-925.
Kurata, S., Go, M. J., Artavanis-Tsakonas, S. and Gehring, W.
J. (2000). Notch signaling and the determination of appendage
identity. Proc. Natl. Acad. Sci. USA
97,2117
-2122.
Lindsley, D. L. and Zimm, G. G. (1992). The Genome of Drosophila melanogaster. San Diego, CA: Academic Press.
Lisowsky, T., Weinstat-Saslow, D. L., Barton, N., Reeders, S. T. and Schneider, M. C. (1995). A new human gene located in the PKD1 region of chromosome 16 is a functional homologue to ERV1 of yeast. Genomics 29,690 -697.[CrossRef][Medline]
Lund, A. H. and van Lohuizen, M. (2004). Polycomb complexes and silencing mechanisms. Curr. Opin. Cell Biol. 16,239 -246.[CrossRef][Medline]
Maves, L. and Schubiger, G. (1995). Wingless
induces transdetermination in developing Drosophila imaginal discs.
Development 121,1263
-1272.
Maves, L. and Schubiger, G. (1998). A molecular
basis for transdetermination in Drosophila imaginal discs: interactions
between wingless and decapentaplegic signaling.
Development 125,115
-124.
Maves, L. and Schubiger, G. (2003). Transdetermination in Drosophila imaginal discs: a model for understanding pluripotency and selector gene maintenance. Curr. Opin. Genet. Dev. 13,472 -479.[CrossRef][Medline]
Merrill, B. J., Gat, U., DasGupta, R. and Fuchs, E.
(2001). Tcf3 and Lef1 regulate lineage differentiation of
multipotent stem cells in skin. Genes Dev.
15,1688
-1705.
Miyoshi, K., Shillingford, J. M., Le Provost, F., Gounari, F.,
Bronson, R., von Boehmer, H., Taketo, M. M., Cardiff, R. D., Hennighausen, L.
and Khazaie, K. (2002). Activation of betacatenin
signaling in differentiated mammary secretory cells induces
transdifferentiation into epidermis and squamous metaplasias. Proc.
Natl. Acad. Sci. USA 99,219
-224.
Niemann, C., Owens, D. M., Hulsken, J., Birchmeier, W. and Watt,
F. M. (2002). Expression of DeltaNLef1 in mouse epidermis
results in differentiation of hair follicles into squamous epidermal cysts and
formation of skin tumours. Development
129,95
-109.
O'Neill, J. W. and Bier, E. (1994). Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. Biotechniques 17, 870, 874-875.[Medline]
Okubo, T. and Hogan, B. L. (2004). Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm. J. Biol. 3,11 .[CrossRef][Medline]
Paro, R., Strutt, H. and Cavalli, G. (1998). Heritable chromatin states induced by the Polycomb and trithorax group genes. Novartis Found. Symp. 214, 51-61; discussion 61-66, 104-113.[Medline]
Paumard-Rigal, S., Zider, A., Vaudin, P. and Silber, J. (1998). Specific interactions between vestigial and scalloped are required to promote wing tissue proliferation in Drosophila melanogaster. Dev. Genes Evol. 208,440 -446.[CrossRef][Medline]
Perez, S. E. and Steller, H. (1996). Molecular and genetic analyses of lama, an evolutionarily conserved gene expressed in the precursors of the Drosophila first optic ganglion. Mech. Dev. 59,11 -27.[CrossRef][Medline]
Petruk, S., Sedkov, Y., Smith, S., Tillib, S., Kraevski, V.,
Nakamura, T., Canaani, E., Croce, C. M. and Mazo, A. (2001).
Trithorax and dCBP acting in a complex to maintain expression of a homeotic
gene. Science 294,1331
-1334.
Pueyo, J. I., Galindo, M. I., Bishop, S. A. and Couso, J. P.
(2000). Proximal-distal leg development in Drosophila requires
the apterous gene and the Lim1 homologue dlim1.
Development 127,5391
-5402.
Raftery, L. A., Sanicola, M., Blackman, R. K. and Gelbart, W. M. (1991). The relationship of decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes mark the anterior-posterior compartment boundary? Development 113, 27-33.[Abstract]
Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R. C. and
Melton, D. A. (2002). `Stemness': transcriptional profiling
of embryonic and adult stem cells. Science
298,597
-600.
Reya, T., Duncan, A. W., Ailles, L., Domen, J., Scherer, D. C., Willert, K., Hintz, L., Nusse, R. and Weissman, I. L. (2003). A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423,409 -414.[CrossRef][Medline]
Ringrose, L., Rehmsmeier, M., Dura, J. M. and Paro, R. (2003). Genome-wide prediction of Polycomb/Trithorax response elements in Drosophila melanogaster. Dev. Cell 5, 759-771.[CrossRef][Medline]
Schubiger, G. (1973). Regeneration of Drosophila melanogaster male leg disc fragments in sugar fed female hosts. Experientia 29,631 -632.[CrossRef][Medline]
Schweizer, P. and Bodenstein, D. (1975). Aging
and its relation to cell growth and differentiation in Drosophila imaginal
discs: developmental response to growth restricting conditions.
Proc. Natl. Acad. Sci. USA
72,4674
-4678.
Simmonds, A. J., Liu, X., Soanes, K. H., Krause, H. M., Irvine,
K. D. and Bell, J. B. (1998). Molecular interactions between
Vestigial and Scalloped promote wing formation in Drosophila. Genes
Dev. 12,3815
-3820.
Sivasankaran, R., Calleja, M., Morata, G. and Basler, K. (2000). The Wingless target gene Dfz3 encodes a new member of the Drosophila Frizzled family. Mech. Dev. 91,427 -431.[CrossRef][Medline]
Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology http://www.bepress.com/sagmb/vol3/iss1/art3
Soto, M. C., Chou, T. B. and Bender, W. (1995).
Comparison of germline mosaics of genes in the Polycomb group of Drosophila
melanogaster. Genetics
140,231
-243.
Strub, S. (1977). Localization of the cells capable of transdetermiantion in a specific region of the male foreleg disc of Drosophila. Wilhelm Roux's Arch. Dev. Biol. 182, 69-74.[CrossRef]
Sustar, A. and Schubiger, G. (2005). A transient cell cycle shift in Drosophila imaginal disc cells precedes multipotency. Cell 120,383 -393.[CrossRef][Medline]
Tobler, H. (1966). Zellspezifische Determination und Beziehung zwischen Proliferation und Transdetermination in Bein- und Flu-gelprimordien von Drosophila melanogaster. J. Embryol. Exp. Morphol. 16,609 -633.[Medline]
Trimarchi, J. M., Fairchild, B., Wen, J. and Lees, J. A.
(2001). The E2F6 transcription factor is a component of the
mammalian Bmi1-containing polycomb complex. Proc. Natl. Acad. Sci.
USA 98,1519
-1524.
Tusher, V. G., Tibshirani, R. and Chu, G.
(2001). Significance analysis of microarrays applied to the
ionizing radiation response. Proc. Natl. Acad. Sci.
USA 98,5116
-5121.
Valk-Lingbeek, M. E., Bruggeman, S. W. and van Lohuizen, M. (2004). Stem cells and cancer; the polycomb connection. Cell 118,409 -418.[CrossRef][Medline]
van de Wetering, M., Cavallo, R., Dooijes, D., van Beest, M., van, Es, J., Loureiro, J., Ypma, A., Hursh, D., Jones, T., Bejsovec, A. et al. (1997). Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88,789 -799.[CrossRef][Medline]
Vervoort, M., Crozatier, M., Valle, D. and Vincent, A. (1999). The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing. Curr. Biol. 9,632 -639.[CrossRef][Medline]
Vierstraete, E., Verleyen, P., Baggerman, G., D'Hertog, W., Van
den Bergh, G., Arckens, L., De Loof, A. and Schoofs, L.
(2004). A proteomic approach for the analysis of instantly
released wound and immune proteins in Drosophila melanogaster hemolymph.
Proc. Natl. Acad. Sci. USA
101,470
-475.
Weaver, T. A. and White, R. A. (1995).
headcase, an imaginal specific gene required for adult morphogenesis in
Drosophila melanogaster. Development
121,4149
-4160.
Williams, J. A., Bell, J. B. and Carroll, S. B. (1991). Control of Drosophila wing and haltere development by the nuclear vestigial gene product. Genes Dev. 5,2481 -2495.[Abstract]
Xu, E. Y., Lee, D. F., Klebes, A., Turek, P. J., Kornberg, T. B.
and Reijo Pera, R. A. (2003). Human BOULE gene rescues
meiotic defects in infertile flies. Hum. Mol. Genet.
12,169
-175.
Yamaguchi, M., Hayashi, Y., Nishimoto, Y., Hirose, F. and
Matsukage, A. (1995). A nucleotide sequence essential for the
function of DRE, a common promoter element for Drosophila DNa
replication-related genes. J. Biol. Chem.
270,15808
-15814.
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