Centro de Biología Molecular, CSIC-UAM, Universidad Autónoma de Madrid, Madrid, 28049, Spain
* Author for correspondence (e-mail: nazpiazu{at}cbm.uam.es)
Accepted 16 November 2004
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SUMMARY |
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Key words: Notum patterning, hth, exd, eyg
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Introduction |
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The dorsal mesothoracic structures are formed by the wing imaginal disc and
include the mesothorax, the wing hinge and the wing blade. The mesothorax is
part of the trunk and is subdivided into an anterior compartment, the notum,
and a posterior one, the postnotum. The notum contains approximately 15,000
cells and is the biggest part of the thorax. It is subdivided by the
pannier (pnr) gene
(Calleja et al., 2000) into
medial and lateral regions, which are in turn further subdivided by the Pax
gene eyegone (eyg) (Aldaz
et al., 2003
).
All the genes involved in these subdivisions encode transcription factors,
and their activities trigger the establishment in the cells of specific
developmental programmes, i.e. states of determination. Experiments in which
these factors are expressed ectopically, which amount to transplantation
experiments, demonstrate that these products are able to induce the formation
of the corresponding pattern out of the normal context
(Aldaz et al., 2003;
Calleja et al., 2000
;
Diez del Corral et al.,
1999
).
Among the genes involved in body patterns, a special case is the pair
homothorax (hth) and extradenticle (exd),
which encode related homeodomain proteins
(Kurant et al., 1998;
Pai et al., 1998
;
Rauskolb et al., 1993
;
Rieckhof et al., 1997
).
exd is expressed ubiquitously, but it is only functional when the Exd
product is transported to the cell nuclei
(Aspland and White, 1997
;
Pai et al., 1998
;
Rieckhof et al., 1997
).
hth is required for the nuclear transport of Exd, and thus acts as a
positive regulator of exd. In turn the Exd nuclear activity is
necessary to prevent the degradation of the Hth product
(Abu-Shaar and Mann, 1998
;
Aspland and White, 1997
;
Mann and Abu-Shaar, 1996
;
Pai et al., 1998
). Thus, the
two gene products are mutually necessary. Besides, the phenotype of
hth and exd mutations is similar or identical
(Abu-Shaar and Mann, 1998
;
Azpiazu and Morata, 2002
;
Kurant et al., 1998
;
Rieckhof et al., 1997
).
Altogether these observations suggest that the two genes are involved in the
same function to which we refer as hth/exd.
A principal role described for hth/exd is its function as cofactor
of Hox products (Peifer and Wieschaus,
1990; Rieckhof et al.,
1997
; Ryoo et al.,
1999
). The Hth and Exd proteins contribute to the specificity of
the Hox products by forming complexes with the different Hox products that
recognise Hox-binding sites with high affinity and specificity
(Chan et al., 1994
;
Ryoo and Mann, 1999
;
Ryoo et al., 1999
;
Van Dijk and Murre, 1994
).
This raises the possibility that Hth and Exd may also interact with other
transcription factors.
In addition to its role as a Hox co-factor, there are hth/exd
functions that seem to be independent of Hox activity. It is involved in the
subdivision of wings and legs into proximal and distal domains
(Abu-Shaar and Mann, 1998;
Azpiazu and Morata, 2000
;
Azpiazu and Morata, 2002
;
Casares and Mann, 2000
;
Gonzalez-Crespo et al., 1998
),
and also has been shown to act as a selector gene in antennal development
(Casares and Mann, 1998
;
Dong et al., 2002
). That
hth/exd may have this kind of function is not totally unexpected as
hth and exd encode transcription factors that may regulate
the transcriptional activity of specific target genes.
The notum of the fly is a region where there is no known Hox gene activity
and is therefore a convenient place to examine possible hth/exd roles
that are not dependent on interactions with Hox genes. Previous results have
indicated that in the absence of exd activity the notum pattern is
abnormal (Gonzalez-Crespo and Morata,
1995), and we have also noticed that the expression of
hth and exd in the notum is not uniform, but shows a
regional modulation. These two observations suggested a Hox-independent
hth/exd function in the notum, which we explore in this report. We
find that hth/exd is necessary to discriminate between two major
parts of the notum, scutum and scutellum, and also contributes to scutellum
identity. We also provide evidence for a molecular interaction between the Hth
and Eyg products.
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Materials and methods |
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For gain-of-function experiments, the following Gal4 lines were used:
ywflp122; act-FRT y+ FRT Gal4 UAS-GFP/SM5 Tb
(Ito et al., 1997),
ap-Gal4 (Rincon-Limas et al.,
2000
), 248-Gal4
(Sanchez et al., 1997
)
eyg-Gal4 (Aldaz et al.,
2003
) and hth-Gal4 (GETDB-Gal4 Enhancer Trap Insertion
Data Base). The gain-of-function clones were generated by recombination at the
FRT sequences. The clones were labelled with Gal4 and GFP
activity. In the adult cuticle the clones can be scored because they are
mutant for f36a and contain y+
activity. The UAS-eyg, UAS-hth and
UAS-tkvQD
(Hoodless et al., 1996
) lines
used were described previously. For the apoptosis experiments we used an
UAS-P35 line (Hay et al.,
1994
). The lacZ reporter line used was the
DC-LacZ (Culi and Modolell,
1998
).
Immunostaining of embryos and discs
Discs were dissected in PBS and fixed in 4% paraformaldehyde for 20 minutes
at room temperature. They were subsequently washed in PBS, blocked in blocking
buffer (PBS, 0.3% Triton, 1% BSA) and incubated overnight with the primary
antibody: anti-ß-Gal 1:2000 (rabbit), anti-Eyg 1:200 (guinea pig),
anti-En 1:10 (mouse), anti-Exd 1:200 (rat), anti-Hth 1:500 (rabbit), anti-Ara
1:200 (rat) and anti-pMad 1:2000 (rabbit) diluted in blocking buffer at
4°C. Washes were performed in blocking buffer, and the appropriate
fluorescent secondary antibody was added for 1 hour at room temperature.
Following further washes in blocking buffer, the discs were mounted in
Vectashield.
Anti-Ara antibody was kindly provided by S. Campuzano, anti pMad by T. Tabata, anti-ß-Gal (rabbit) and anti-En were purchased from Cappel and from the Hybridoma Bank respectively. Images were taken in a laser MicroRadiance microscope (BioRad) and subsequently processed using Adobe Photoshop.
Protein interaction experiments
Crude Drosophila discs extracts were prepared by homogenizing 0.2
ml of third instar larvae in 0.4 ml of lysis buffer (1xPBS, 1% NP-40, 1
mM PMSF and 20 µg/ml each of peptastin and leupeptin). The homogenates were
centrifuged, and the aqueous phase was mixed with the His-tagged, full-length
Hth protein extracted under native conditions. The complexes formed were
purified using the Ni-NTA Agarose (Quiagen), washed five times in 1xPBS,
0.5 M NaCl and one final time in 50 mM Tris (pH 6.8). They were boiled in
loading buffer and resolved by SDS-PAGE. After blotting to nitrocellulose, the
filter was incubated with the anti-Eyg antibody and the signal was detected
using the Amersham ECL western blotting analysis system.
Preparation of larval and adult cuticles
For good X-gal staining of adult patterns, pharates were removed from the
puparium and treated as described by Calleja et al.
(Calleja et al., 1996). To
examine the cuticular patterns of the different genetic combinations adult
flies were prepared by the standard methods for microscopic inspection. Soft
parts were digested with 10% KOH, washed with alcohol and mounted in Euparal.
Embryos were collected overnight and aged an additional 12 hours. First instar
larvae were dechorionated in commercial bleach for 3 minutes and the vitelline
membrane removed using heptano-methanol 1:1. Then, after washing with methanol
and 0.1% Triton X-100, larvae were mounted in Hoyer's lactic acid 1:1 and
allowed to clear at 65°C for at least 24 hours.
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Results |
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We have further delimited the expression of hth with respect to
that of other developmental genes that are active in the notum. Double
staining with antibodies directed against the En, Eyg and pMad
(Tanimoto et al., 2000)
products is shown in Fig. 1E-G.
Of special interest is the comparison of the eyg and hth
domains (Fig. 1F): the
subdomain of hth is included within the eyg domain, but the
ß subdomain abuts in its anterior border with the posterior border of
eyg. This subdomain coincides with high levels of pMad
(Fig. 1G), indicating that the
Dpp signalling pathway is active in this domain. X-gal staining of
hth-Gal4/UASlacZ flies (Fig.
1I) shows that the hth
subdomain differentiates
the anterior lateral part of the adult notum and the ß subdomain the
scutellar region. It is also of interest to point out that the onset of
hth modulation at early third instar
(Fig. 1A) correlates with the
initiation of eyg activity, which occurs approximately at that time
(Aldaz et al., 2003
).
Developmental function of hth/exd in the notum
We have studied the developmental role of hth/exd in the notum by
examining the consequences of the loss of function and of modifying its normal
spatial expression.
Notum development in the absence of hth/exd function
Null hth and exd mutants are zygotic lethal, therefore we
have induced marked clones to study the behaviour of mutant cells in imaginal
discs and the adult patterns they form. As their phenotype is the same, we
have used indistinctly exd or hth mutant clones as
convenient. For the adult cuticle we have used exd clones as they are
better marked for the adult notum structures (see Materials and methods). We
made use of the Minute technique (Morata
and Ripoll, 1975) to induce large clones. Some of those that
initiated early in development occupy a large part of the notum thus allowing
the visualisation of the pattern produced in absence of hth/exd
function (Fig. 2). The results
obtained after having examined more than hundred clones can be summarised by
saying that the loss of hth/exd does not produce a change of identity
of the notum cells but gives rise to an abnormal notum pattern. It is
illustrated in the cartoon of Fig.
2C, where we compare a normal notum pattern with that formed by
the composite of a number of exd mutant clones. There are
supernumerary macrochaetes on the presutural area
(Fig. 2A), the infrascutellar
suture disappears and the postalar bristles are not formed. The suture between
scutum and scutellum also disappears (Fig.
2B), and the more posterior bristles tend to align along with the
dorsocentral ones. Moreover, these clones often differentiate microchaetes in
the scutellum region.
|
Notum development with uniform hth/exd function
The previous experiment tested the functional role of hth/exd in
the notum pattern. We then tried to assess the functional significance of the
modulation of hth/exd activity. We have used the Gal4/UAS method to
replace the normal expression pattern by a uniform pattern as driven by the
Gal4 lines ap-Gal4 and 248-Gal4. As shown in
Fig. 3A, the notum region of an
ap-Gal4>UAS-hth wing disc presents an uniform hth
activity, very different from the normal pattern-compare with
Fig. 1B.
|
The size reduction observed in these flies seems not to be a consequence of massive apoptosis. We have checked for levels of active caspase in discs of genotype ap-Gal>UAS-hth or 248-Gal>/UAS-hth and did not find a significant increase compared with wild-type discs. Moreover, we were not able to restore the normal size of the notum in flies of those genotypes by co-expressing the caspase inhibitor P35 (not shown).
The conclusion from the previous experiments is that hth/exd is not involved in fate specification of the notum cells: in the absence of hth/exd function the cells still differentiate notum pattern elements. However, the local modulation of hth/exd expression appears to be an important factor in the patterning process.
Regulation of hth expression
It follows from the results above that the understanding of the spatial
regulation of hth/exd may provide insights into the notum patterning.
As the hth expression in the notum evolves from ubiquitous to
spatially restricted during the larval period, one of our aims was to identify
the factors responsible for this regulation.
eyg is a negative regulator of hth
We suspected that eyg might regulate hth/exd because, as
shown in Fig. 1F, hth
and eyg expressions abut in some places as if they were mutually
exclusive. In particular, the anterior border of the hth ß
subdomain abuts with the posterior border of the eyg domain.
We first examined hth expression in eygSA2
homozygous discs. As shown in Fig.
4A it becomes uniform and covers most of the notum, suggesting
that in absence of Eyg there is an expansion of the hth ß
subdomain. This result indicates that eyg can function as a negative
regulator of hth/exd. However, in normal development the hth
subdomain is included within the eyg domain
(Fig. 1F), suggesting that the
repressive role of Eyg is effective only in part of the domain. To explore
this possibility further, we induced clones of mutant eyg cells all
over the notum and examined their local effect on hth/exd expression.
We obtained two types of eyg- clones in respect of their
effect on hth expression. Those in the
subdomain, where
hth and eyg are normally co-extensive, have no effect on
hth expression (not shown). However, the clones in the posterior
region of the eyg domain, located between the
and ß
hth subdomains (Fig.
4B,C) show ectopic hth activity. This result indicates
that eyg acts as a negative regulator of hth in the
posterior region of its domain; the sharp border of hth in the
posterior region probably reflects the repression by eyg. We have
examined eyg expression in hth- clones (not
shown) and found that it is not modified.
|
The Dpp pathway is a negative regulator of hth
Because high levels of Dpp signalling co-express with the ß
hth subdomain, we wanted to determine whether the Dpp pathway could
be involved in hth regulation. We generated clones of cells mutant
for Mothers against dpp (Mad), which are unable to transduce
the Dpp signal (Newfeld et al.,
1996), and examined them for hth expression.
According to their position Mad- clones have different
effects on hth expression (Fig.
5): (1) those located in the subdomain do not affect
hth; (2) those located between the
and ß subdomains
exhibit gain of hth; and (3) the clones in the ß subdomain lose
hth expression. The loss of hth by the
Mad- clones in the ß subdomain appears to be an
indirect consequence of the up regulation of eyg in absence of
activity of the Dpp pathway (Aldaz et al.,
2003
). As we show above, the Eyg product represses
hth/exd in this region.
|
To confirm the repressor role of Dpp, we generated clones of cells that
contain TkvQD a constitutive form of the Dpp receptor, which
produces a hyperactivation of the Dpp pathway
(Nellen et al., 1996). The
results are illustrated in Fig.
5D-F: TkvQD clones repress hth when they are
generated in the
subdomain, but have no effect in the ß
subdomain.
In summary, from the preceding experiments, both Eyg and the Dpp pathway
act as repressors of hth in the inter-subdomains region. However, in
both cases their activity domains are co-extensive in part with the
hth domain; the hth subdomain for Eyg and the
hth ß subdomain for the Dpp pathway. This indicates that Eyg and
the Dpp pathway require additional factors to fulfil its repressing role (see
Discussion).
Interactions between the Hth and the Eyg proteins
The and the ß subdomains differentiate distinct patterns in
the adult notum (Fig. 1). The
subdomain, where hth/exd and eyg are co-extensive,
gives rise to a part of the scutum and contains microchaetes as a distinctive
feature. The ß subdomain, which does not contain eyg activity,
differentiates the scutellum and part of the lateral region, which do not
contain microchaetes. This suggests that the differential expression of
hth/exd and eyg may contribute to the pattern differences.
To check on this possibility, we have expressed the Hth and Eyg products under
the control of the same driver (248-Gal4), both separately and
together, and compared the notum patterns obtained. In
248-Gal4>UAS-hth, we observe
(Fig. 6A) a reduction of the
notum, which affects mostly the scutum territory; as a result, it
differentiates into very few microchaetes and the dorsocentral macrochaetes
are missing. We have checked in discs of this genotype the expression of the
specific dorsocentral enhancer of scute
(Culí and Modolell,
1998
) and found it is not expressed. In
248-Gal4>UAS-eyg (Fig.
6B), there is a mirror image duplication of the scutum pattern in
the scutellum, as reported previously
(Aldaz et al., 2003
). This
duplication includes macrochaetes, which are part of the eyg domain
(Aldaz et al., 2003
). By
comparison, the duplicated notum structures in the 248-Gal4>UAS-hth
UAS-eyg genotype do not contain macrochaetes, suggesting that the
duplicated pattern corresponds to the
subdomain. This interpretation
is supported by the observation that in discs of this genotype the DC enhancer
of scute is not expressed (not shown).
|
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Discussion |
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Additionally, hth/exd has some other roles that are not directly
related to Hox gene activity. During limb development, it acts to repress the
response of the proximal region to the influence of Hh/Wg/Dpp pathways, thus
contributing to the subdivision of appendages along the proximodistal axis
(Abu-Shaar and Mann, 1998;
Azpiazu and Morata, 2000
;
Azpiazu and Morata, 2002
;
Gonzalez-Crespo et al., 1998
).
This function is also conserved in vertebrate development
(Mercader et al., 1999
).
Here, we deal with a novel hth/exd function: its patterning role
in the notum. It is not related to the specification of notum identity because
it is not affected by alterations of hth/exd activity. For example,
in the absence of hth/exd the cells still differentiate as notum, if
an abnormal one (Fig. 2). Conversely, high and uniform Hth levels also produce notum tissue but with
abnormal pattern (Fig. 3). This
function is only required in part of the notum and is therefore linked to the
modulation of hth expression during the development of the disc. The
final result of this modulation is the appearance of the and ß
subdomains of hth that we report here. These two subdomains
differentiate distinct notum patterns, suggesting that Hth/Exd interact with
other localised products to generate these patterns.
Thus, there are two principal aspects in the patterning function of
hth/exd: (1) the spatial regulation, that eventually results in the
restriction of its expression to the and ß subdomains; and (2)
the local interactions of Hth/Exd with other products in either of the
subdomains.
Regulation of hth/exd in the notum
Although hth and exd form a single functional unit, their
mode of regulation is different: exd is expressed ubiquitously but is
regulated at the subcellular level by hth, which promotes Exd nuclear
transport. Therefore, the key element of hth/exd regulation is the
transcriptional control of hth.
Originally, hth is expressed in all the notum cells
(Azpiazu and Morata, 2002)
(this report) and later becomes restricted to the
and ß
subdomains. Consequently, the principal aspect of hth regulation is
the mechanism(s) leading to its repression in the regions outside the
and ß subdomains. We have identified two negative regulators: the
eyg gene and the Dpp pathway, which probably acts through some
unidentified downstream gene. In the notum hth behaves as a
downstream target of both the Dpp pathway and eyg.
The role of Eyg as a negative regulator of hth is based on the
following observations: (1) the beginning of the modulation of hth
expression in the notum at the early third instar coincides with the
initiation of eyg expression
(Aldaz et al., 2003); (2) in
eyg mutants the hth domain is expanded, extending to most of
the notum (Fig. 4A); (3) mutant
eyg clones show hth derepression in the inter-subdomains
region (Fig. 4B,C), and
conversely, ectopic eyg activity in the ß subdomain represses
hth (Fig. 4). The fact
that it fails to affect hth in the
subdomain was expected as
eyg and hth are normally co-expressed in this subdomain
(Fig. 1F). In conclusion,
eyg suppresses hth in the inter-subdomains region and also
acts as a barrier for hth in the eyg/ß-hth
border.
The role of the Dpp pathway as a negative regulator of hth is
based on results illustrated in Fig.
5A-C, showing that Mad- mutant clones in the
inter-subdomains region show activation of hth. This is in contrast
to the behaviour of those clones in the subdomain, where they have no
effect, or in the ß subdomain, where they show suppression of
hth. We believe that the reason for the latter effect is that
eyg is up regulated in those clones, as described previously
(Aldaz et al., 2003
), and in
turn Eyg suppresses hth, as we discuss above. The lack of effect of
Mad- clones in the
subdomain is probably due to
the low activity of Dpp in that region (see below). The observation
(Fig. 5D-F) that the high
activity levels generated in the TkvQD clones suppress hth
in this subdomain in principle supports this view. Expectedly,
TkvQD clones do not affect hth expression in the ß
subdomain, because it normally possesses high Dpp activity levels.
Taking all the results together, we propose the following model of
hth regulation (Fig.
7). As hth is originally expressed in all trunk embryonic
cells (Azpiazu and Morata, 1998) and in all the notum cells in the early disc
(Azpiazu and Morata, 2002), the
regulation of hth during wing disc development essentially reflects
local repression in specific parts of the disc. The basic idea is that
hth is repressed by the joint contribution of eyg and
high/moderate levels of the Dpp pathway. Neither of these elements can repress
hth individually. Although eyg appears to act uniformly in
its domain, the repressing activity of Dpp is concentration dependent. Within
the eyg domain, the hth
subdomain is located in the
anterior region, in which the Dpp levels are too low to be effective and Eyg
alone cannot repress hth/exd. In the inter-subdomains region the Dpp
levels are high enough to repress hth as here it acts together with
Eyg. The ß subdomain is outside the eyg domain and therefore in
the absence of Eyg even the high Dpp levels are not capable of repressing
hth/exd. The model is also supported by the experiments of
overexpressing eyg. The eyg-expressing clones in the ß
subdomain suppress hth because the two repressors are active in the
clones, while they have no effect in the
subdomain because it normally
contains high eyg levels. In principle the experiments overexpressing
the Dpp pathway (TkvQD clones) appear to support the model. These
clones have no effect in the ß subdomain, which normally possesses high
Dpp activity levels, but they suppress hth in the
subdomain.
However, these clones are known to suppress eyg
(Aldaz et al., 2003
) and
therefore hth should not be repressed according to our model. It is
possible that in certain circumstances the very high Dpp activity levels
induced by these clones may be sufficient to down regulate hth, even
in the absence of eyg.
|
Interactions of Hth/Exd with other products
The second aspect of the late patterning function of hth/exd
arises from the observation that the and ß subdomains form
different patterns with similar levels of hth. This suggests the
existence of interactions between Hth/Exd and products specifically localised
to the different subdomains. In the case of the
subdomain, the obvious
candidate for the interaction is Eyg. We show that the joint activity of
hth/exd and eyg specifies a notum pattern that is different
from those specified by each of these genes alone
(Fig. 6A-C).
Our finding that the Eyg and Hth proteins associate to form a complex in
vitro suggest a mechanism to achieve the pattern difference between the
and the ß subdomains. As it has been shown to be the case for the
in vivo specificity of the Hox genes, the association of Hth/Exd with the
different Hox products results in higher affinity and specificity for target
sites (Ryoo et al., 1999
).
Here, the formation of a Eyg/Hth/Exd complex in the
subdomain may
result in a constellation of gene activity different from that in the ß
subdomain where Eyg is not present. In the latter subdomain hth/exd
may act alone, for after all the two genes encode transcription factors.
Alternatively, the Hth/Exd products may interact with some other yet
unidentified co-factor.
Genetic subdivisions of the notum
As pointed out in the Introduction, the morphological diversity of the body
is achieved by regulating the spatial expression of developmental regulatory
genes. For example, the spatial deployment of the various Hox genes along the
anteroposterior body axis establishes the identity of the different segments
(reviewed by Lawrence and Morata,
1994; Mann and Morata,
2000
). Within each segment, this process is reiterated to
elaborate the identity of the anterior or posterior compartments, which
requires the deployment of engrailed
(Morata and Lawrence, 1975
).
Further genetic subdivisions establish the identity of even more discrete
regions. In the notum, previous work has identified two subdivisions that
appear during the development of the disc. One is established by pnr,
which subdivides the notum into a medial and a lateral region
(Calleja et al., 2000
). The
second results from the activation of the eyg gene and straddles the
pnr domain, thus originating four genetically distinct regions
(Aldaz et al., 2003
). Here, we
report a new element involved in the notum subdivision: the appearance of
hth expression in two distinct subdomains. These two hth
subdomains are superimposed with the genetic subdivisions established by
en, pnr and eyg, and contribute to the genetic
diversification of the notum and hence to the morphological diversity within
it. It is part of a genetic address
(Garcia-Bellido et al., 1979
)
that specifies the final pattern. As shown in
Fig. 6C, the combination of Hth
with Eyg produces a notum pattern different from that produced by Hth alone
(or in combination with some unknown factor). Our demonstration that the Hth
and the Eyg products form a protein complex suggests that protein-protein
interactions are part of the patterning process.
One interesting aspect of the interaction of hth/exd and eyg is that it acts in two different ways. At the gene regulation level, eyg participates in the spatial control of hth/exd activity, but where the two genes are co-expressed their proteins interact, presumably to contribute to the in vivo affinity and specificity for target genes.
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ACKNOWLEDGMENTS |
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