Institut für Zoophysiologie der Universität Bonn, Abt. für
Entwicklungsbiologie, Poppelsdorfer Schloss, 53115 Bonn, Germany
* These authors contributed equally to this work
Author for correspondence (e-mail:
m.hoch{at}uni-bonn.de
)
Accepted 12 February 2002
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Summary |
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Key words: Gap junctions, Wingless signalling, innexin 2, Proventriculus, Drosophila
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Introduction |
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Another class of molecules that is involved in the direct communication
between neighbouring cells are transmembrane channel proteins that assemble
into gap junctions (for reviews, see Kumar
and Gilula, 1996; Goodenough
et al., 1996
). Gap junctions are composed of two hexameric
hemichannel subunits that are transported to the plasma membrane, following
the secretory pathway for transmembrane proteins (for reviews, see
Falk and Gilula, 1994
;
Zhang et al., 1996
;
Yeager et al., 1998
). If
hemichannels of adjacent cells interact, they form a functional dodecameric
gap junction channel directly linking cytoplasm of neighbouring cells
(Unger et al., 1997
). This
allows cells to exchange ions and small molecules that can participate in
signalling events. Gap junctions are clusters of intercellular channels and
may consist of tens or thousands of channels forming so-called plaques in the
membranes of cells. It is believed that the permeability of the intercellular
channels for ions and small molecules depends on their molecular composition
and on the size and charge of the permeant molecules (for reviews, see
Kumar and Gilula, 1996
;
Goodenough et al., 1996
). How
plaque formation and localisation is controlled during developmental processes
is largely unknown. In vertebrates, gap junction channel proteins are coded by
the connexin multigene family (Willecke, 1991). Many tissues express several
different connexin isoforms with complex and overlapping temporal and spatial
profiles. It has been shown that heteromeric channels can occur in vivo and
that heterotypic ones can be formed in vitro, suggesting that the normal
physiological functioning of cells may require expression of multiple
connexins (for a review, see Yeager et
al., 1998
). In invertebrates, a separate gene family encoding
gapjunction channel proteins has been identified, the innexin
multigene family (Phelan et al.,
1998a
) (for review, see Phelan
and Starich, 2001
). Innexins are structurally and functionally
analogous to connexins; however, they share no sequence homology (for a
review, see Phelan and Starich,
2001
). Electrophysiological evidence obtained in paired
Xenopus oocytes suggests that innexins, like connexins, form
heteromeric channels (Landesman et al.,
1999
; Phelan et al.,
1998b
; Stebbings et al.,
2000
). The innexin mutants that have been identified in
C. elegans and Drosophila display defects in electrical
signal transmission. Mutations in the C. elegans unc-7 and
unc-9 genes cause uncoordinated phenotypes
(Starich et al., 1996
;
Barnes and Hekimi, 1997
), and
eat-5 mutants display an impairment of electrical coupling between
pharynx muscles (Starich et al.,
1996
). In Drosophila, shaking-B mutants are characterised
by a loss of electrical signalling at synapses of the giant fibre system
(Phelan et al., 1996
), and
ogre mutants display a reduced optic lobe primordium (Watanabe et
al., 1990). Most of the six other known Drosophila innexin genes
(Adams et al., 2000
) are
expressed in a complex and overlapping temporal and spatial profile, with
several members showing high levels of expression in the developing embryonic
gut (Curtin et al., 1999
;
Stebbings et al., 2000
).
In a screen of P-element insertion lines to identify novel regulators of gut development, we isolated four P alleles of a locus that we named kropf. The mutant phenotype of the different P alleles is highly similar. Most of the mutant animals are embryonic lethal; however, a few mutants reach the first instar larval stage and display a feeding defect. Molecular analysis has revealed that the kropf locus corresponds to the innexin 2 transcription unit and that at least two P alleles are innexin 2 transcript null alleles. Expression studies indicate that innexin 2 is expressed maternally and zygotically. We now report that the feeding defect of kropf mutant larvae is due to a malformation of the proventriculus, an organ that controls food passage at the foregut/midgut boundary of the larval gastrointestinal tract. It originates during embryogenesis by the migration and subsequent folding of ectodermal and endodermal tissue layers. In kropf mutants, the formation of the keyhole, a primordial structure that was shown to be generated by the Wingless- and Hedgehog-dependent outfolding, and subsequent migration of proventriculus primordial cells, does not occur properly. Genetic lack- and gain-of-function studies and experiments in Drosophila tissue culture cells demonstrate that innexin 2 transcription is induced by Wingless signalling in the foregut.
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Materials and Methods |
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Molecular characterisation of the kropf P alleles
Genomic DNA flanking the P-element insertions was recovered by plasmid
rescue and inverse PCR. The precise location of the P-element insertion was
determined by sequencing the rescued DNA and the amplified fragments. The
P-element insertion sites are kropfP16 +236,
kropfP34 -11, kropfP36 +197 and
kropfP188 +188, with respect to the transcription start
site at position +1 of the innexin 2 gene
(Stebbings et al., 2000).
Feeding assay
To monitor larval feeding behaviour, the animals were allowed to grow on
grape agar plates and we fed them yeast that was dyed with Carmine Red (Sigma)
(Pankratz and Hoch, 1995).
Phenotypes were scored at various times under the dissecting microscope.
Antibody staining
The following antibodies were used: anti-Wingless (1:20 gift of S. Cohen),
anti-Dve (1:1000; gift of F. Matsuzaki), anti-Fkh (1:100, gift of H.
Jäckle) and mouse anti-ß-Gal (1:1000; Developmental Studies
Hybridoma Bank). For immunostaining, embryos were collected and processed
following standard procedures (Fuß
et al., 2001), incubated overnight at 4°C in primary antibody
mix containing preabsorbed antibody. Biotinylated secondary antibodies,
Vectastain and DAB staining reagents were used according to manufacturer's
recommendations (Vector Laboratories). For fluorescent microscopy, secondary
antibodies Alexa Fluor488 and Alexa Fluor546 from
MoBiTec were used at dilutions of 1:200 and 1:400, respectively.
In situ hybridisation
For in situ hybridisation, digoxigenin- or fluorescein-labelled RNA
antisense probes were generated by in vitro transcription of a C-terminal and
full-length innexin 2 fragment from the innexin 2 cDNA clone
LD clone 11658 (Berkeley Genome Project), according to the manufacturer's
instructions (Roche, Mannheim). Fluorescent detection of innexin 2
transcripts, co-immunostained with anti-Discs lost antibody, which marks the
apical sides of epithelial cells (Bhat et
al., 1999), was performed as described previously
(Hughes et al., 1996
;
Hughes and Krause, 1999
).
Rabbit anti-Discs lost and mouse anti-Fluorescein antibodies were used at
dilutions of 1:750 and 1:2000, respectively. As secondary antibodies, we used
Cy2-conjugated anti rabbit antibody (1: 200; Dianova) and Cy3-conjugated anti
mouse antibody (1:400; Dianova). Images were obtained using a Leica DM IRB
laser-scanning confocal microscope. Each fluorochrome was scanned individually
to avoid crosstalk between channels. Images were subsequently combined using
Adobe Photoshop 5.1.
Isolation and reverse transcription (RT)-PCR analysis in
Drosophila S2 cells
Total RNA from Drosophila Schneider cells that were either
transfected with the pIB expression vector alone or with pIBArm, which
contains the Armadillo cDNA, was isolated using the Qiagen RNeasy Mini Kit,
according the manufacturer's instructions (Qiagen). For RT-PCR analysis, 2
µg of DNase I-treated total RNA was reverse transcribed by using 25 U of
avian murine virus reverse transcriptase (Roche, Mannheim) with an innexin
2-specific antisense primer. The reaction mixture contained total RNA,
5xreverse transcriptase buffer provided by the manufacturer, 35 U of
RNase inhibitor (Pharmacia Biotech) and 0.2 mM of deoxynucleoside
triphosphates (dNTP) in a final volume of 30 µl. The mixture was heated to
65°C for 5 minutes. AMV reverse transcriptase was added, and the mixture
was incubated at 42°C for 60 minutes. After the completion of the
first-strand cDNA synthesis, one-third of the reaction volume was amplified
directly by using 5 U of Taq polymerase and 5 mM of innexin
2-specific antisense (5'-TTAGGCGTCGAAGGGCCGCTT-3') and sense
primers (5'-CTGAGCATCATGTCGGGAATATCGC-3'). PCR was performed in a
final volume of 50 µl by using the Roche `High Fidelity PCR-kit'. The
predicted PCR product was 273 bp. The predicted size for the actin
amplification product (5'-AGCCAGCAGTCGTCTAATCCAG-3' and
5'-CAGCAACTTTCTTCGTCACACAT-3'), which was used as an internal
control, is 210 bp.
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Results |
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The four kropfP alleles all showed very similar mutant phenotypes. About two-thirds of the mutant animals were embryonic lethals; however, about one-third of the mutants died in the first instar larval stage (see Materials and Methods). As the P lines showed expression patterns in the developing proventriculus, we tested kropfP larvae for their ability to feed dye-coloured yeast. We had shown previously that malformation of the proventriculus results in a feeding defect causing larval lethality (Hoch and Pankratz, 1995). Using this feeding assay, we found that in kropf mutant larvae, the food could not be efficiently transported into the midgut, resulting in an engorged oesophagus. Ectodermal cells of the foregut which in wild-type larvae migrate into the endodermal proventricular pouch tissue to form the multi-layered proventriculus organ (Fig. 2A, magnification in C), fail to move inwards in the mutant animals and are stuck on top of the endoderm cells (Fig. 2B, magnification in D). We observe lethality of the mutant larvae in the first instar stage, indicating that the feeding defect may result in death by starvation. For further mutant analysis, we used the l(1)G0016 (kropf P16) allele.
|
Innexin 2 is required for keyhole formation during proventriculus
development
The proventriculus is an organ that functions as a valve to regulate food
passage from the foregut into the midgut in Drosophila larvae
(Snodgrass, 1935;
Strasburger, 1932
). It
develops during early embryogenesis at the junction of the ectodermal foregut
and the endodermal anterior midgut primordia
(Skaer, 1993
;
Fuß and Hoch, 1998
;
Nakagoshi et al., 1998
). There
is initially an outward buckling of the ectoderm, in a region that is referred
to as the `keyhole' structure (Pankratz
and Hoch, 1995
). This area will undergo further outward movement,
then fold back on itself and move inwards into the endodermal proventricular
pouch to form the mature, multi-layered proventriculus
(King, 1988
). Wingless is
functionally required for cell migration of the proventricular cells and is
initially expressed in wild-type embryos in a domain that includes the region
from which the keyhole will form (Pankratz
and Hoch, 1995
; Fuß and
Hoch, 1998
) (Fig.
3A, magnification in C). With the onset of cell migration during
keyhole formation, Wingless expression is either lost or repressed in the
centre of its expression domain resulting into two domains that are split
(Fig. 3B, magnification in D).
One domain is at the anterior border of the keyhole in the ectodermal foregut
cells and the other in endodermal cells posterior to the keyhole. In kropf
P mutants, initial Wingless expression in the keyhole cells is normal
(Fig. 3E, magnification in G).
However, the Wingless expression in the central region persists
(Fig. 3F, magnification in H).
The cells that would normally form the keyhole stay fixed within the tightly
organised epithelial foregut tube in the mutant until late stages of
development, and the migration of the ectodermal cells into the endodermal
pouch seems not to occur. Instead, these cells stay on top of the endoderm, as
can be demonstrated using by anti-Fkh/anti-Dve double staining
(Fig. 3I,J). We conclude from
these results that innexin 2 is required during early stages of
proventriculus development for keyhole formation. In kropf mutants,
the keyhole defect prevents the correct folding and inward movement of the
proventriculus tissue layers most probably resulting in the feeding defect
observed later in the mutant larvae.
|
Apical Innexin 2 mRNA localisation
In order to analyse the role of innexin 2 during development
further, we studied the mRNA expression pattern of the gene using an
innexin 2 antisense probe (see Materials and Methods). During
oogenesis, innexin 2 transcripts are found in the nurse cells, border
cells and in a cortical localisation in the oocyte
(Fig. 4A), consistent with a
maternal complement that could explain the variability of the kropf
mutant phenotype. innexin 2 mRNA is ubiquitously distributed during
the early embryonic blastoderm stage (Fig.
4B). A segmental expression pattern is found during germ band
extension stage (Fig. 4C)
(Stebbings et al., 2000) and
we find expression in the stomodeal and proctodeal invaginations, which are
the primordia of the foregut and the hindgut. During keyhole formation at the
boundary of the foregut and the anterior midgut, innexin 2 expression
persists in the ectodermal and endodermal tissue regions of the proventriculus
primordium (Fig. 4D). During
the invagination of the ectodermal cells into the endodermal pouch, the
expression pattern of innexin 2 is restricted to the endodermal part
of the proventriculus (not shown). Fluorescent detection of innexin 2
transcripts (Materials and Methods) and co-immunostaining with anti-Discs lost
antibody, which marks the apical sides of epithelial cells
(Bhat et al., 1999
)
demonstrates that innexin 2 mRNA is localised to the apical sides of
the proventricular cells (Fig.
4E-G). In summary, our mRNA expression studies are compatible with
a functional role of innexin 2 during proventriculus development.
Innexin 2 is a target gene of the Wingless signalling pathway
Morphogenetic cell movements of the proventriculus epithelium and of the
hindgut depend on the activities of the genes hedgehog, wingless and
decapentaplegic (Pankratz and
Hoch, 1995; Hoch and Pankratz,
1996
). Through the restricted expression of these genes in local
domains, they define signalling centres at the foregut/midgut and the
midgut/hindgut boundaries. Wingless and Hedgehog were shown to control cell
migration of epithelial gut cells whereas Decapentaplegic was shown to prevent
morphogenesis in the gut (Pankratz and
Hoch, 1995
; Hoch and Pankratz,
1996
). In order to analyse whether innexin 2
transcription is dependent on these signalling cascades, we studied the
transcriptional regulation of the gene by crossing the P lines into various
mutant backgrounds or by determining the innexin 2 mRNA profile (the
nuclear ß-Gal expression pattern reflects the mRNA expression profile of
innexin 2 during proventriculus development). Whereas in
hedgehog and decapentaplegic mutant embryos, innexin
2 or reporter gene expression was not affected, they were absent in the
proventriculus rudiment and reduced in the small intestine region of amorphic
wingless mutants (Fig.
5A,B). Because the gut morphology in general is strongly affected
in wingless mutants, we also performed a gain-of-function analysis
using the UAS-Gal4 system (Brand and
Perrimon, 1993
). Ectopic expression of Wingless in the visceral
mesoderm using the twi-Gal4 driver in combination with the
UAS-wingless effector line or ectopic expression in the endoderm
using the 14-3fkh-Gal4 driver in combination with the UAS-wingless
effector, resulted in ectopic expression of innexin 2 or ß-Gal
expression in the anterior and posterior regions of the gut
(Fig. 5C,D). Both, the
expression domains in the proventriculus and in the small intestine region
that lies at the complementary endoderm/ectoderm boundary in the posterior
region, dramatically expand (Fig.
5D). It is known that upon ectopic Wingless expression, endogenous
decapentaplegic expression is ectopically activated and covers the
region of parasegments 2-7 of the visceral mesoderm
(Yu et al., 1996
). However,
upon ubiquitous expression of Decapentaplegic in the visceral mesoderm using
UAS-decapentaplegic transgenes, in combination with twist-Gal4, we
could not find a similar alteration of the innexin 2 expression
domains.
|
To further substantiate the conclusion drawn from our genetic experiments
that innexin 2 transcription is regulated in response to Wingless
signalling, we performed transfection experiments in Drosophila
tissue culture S2 cells (see Materials and Methods). Total RNA was isolated
from Schneider S2 cells that were either transfected with an expression vector
alone or with the same vector containing the cDNA of Armadillo, the
ß-catenin homologue that has been shown to serve as the transducer of the
Wingless signalling cascade (Peifer and
Polakis, 2000). We monitored innexin 2 mRNA levels by
performing a RT-PCR analysis using actin mRNA levels as an internal control
(see Materials and Methods). As shown in
Fig. 5E, innexin 2
mRNA levels are increased approximately five times in response to Armadillo
when compared with the control reaction, whereas the actin mRNA levels remain
constant in both situations. We conclude from our genetic lack- and
gain-of-function studies, and from our tissue culture experiments that
innexin 2 is a target gene of the Wingless signalling cascade that is
induced in wild-type embryos at ectoderm/endoderm boundaries during gut
organogenesis (Fig. 5F).
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Discussion |
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Innexin 2 is required for keyhole formation
Our results indicate that kropf mutant larvae which are devoid of
innexin 2 expression, display a feeding defect resulting from a
failure of proventriculus fomation in the embryo: the ectodermal
proventriculus cells fail to move into the endodermal pouch and are stuck
instead on top of the endoderm tissue; a multi-layered organ does not form
(Fig. 2). Our mutant analysis
using Wingless and Fkh/Dve as markers to visualise the ectodermal/endodermal
boundaries in the foregut strongly suggests that this late invagination defect
originates from a failure of cells to form the keyhole structure at much
earlier stages (Fig. 3). The
keyhole is the first visible morphological structure that is formed by an
outbuckling of primordial cells of the proventriculus, in a region of the
caudal foregut primordium that is free of visceral mesoderm
(Pankratz and Hoch, 1995).
innexin 2 mRNA is expressed in the primordial cells prior to the
initiation of keyhole formation (Fig.
4). The analysis of Wingless expression in the proventriculus
primordium of kropf mutants suggests that it is not the specification
of the keyhole cells that is affected in the mutant. At the time when the
keyhole forms, cell proliferation in the foregut has already been completed
(Campos-Ortega and Hartenstein,
1997
). Furthermore, we could not observe cell death of keyhole
cells in kropf mutants using TUNEL analysis (data not shown). We
therefore favour a model that suggests that gap junctional communication may
be important for the cellular processes involved in the outbuckling, the
folding and the subsequent migration of the keyhole cells, as has been
observed previously for the migration of neural crest cells in vertebrates
(Lo et al., 1999
;
Huang et al., 1998
).
Innexin 2 is activated by Wingless signalling at the
ectoderm/endoderm boundaries of the intestinal canal
Epistasis experiments indicate that innexin 2 transcription is
abolished in the proventriculus rudiment that is left in wingless
mutants and that expression of the gene is dramatically expanded upon ectopic
expression of Wingless in the gain-of-function experiment
(Fig. 5). Furthermore
Armadillo, the signal transducer of the Wingless signalling pathway is able to
induce innexin 2 transcription in tissue culture cells, as show by
RT-PCR experiments (Fig. 5E).
In wild-type embryos, both innexin 2 and wingless are
co-expressed during early and late stages of proventriculus development
(Fig. 3A,C, compare to
Fig. 4D). In summary, these
data provide strong evidence that the gap junction channel gene innexin
2 is induced as a Wingless target gene during keyhole formation in the
proventriculus primordium. Recently, defective proventriculus
(dve), a novel homeobox gene, has been identified as another target
gene of Wingless signalling in the proventriculus endoderm
(Fuß and Hoch, 1998;
Nakagoshi et al., 1998
).
dve mutants display, however, a late invagination defect caused by a
malformation of the proventriculus endoderm
(Fuß and Hoch, 1998
;
Nakagoshi et al., 1998
). This
suggests that Wingless has the potential to activate target genes that act
during different phases of proventriculus development. innexin 2
expression is, however, not only regulated by Wingless in the proventriculus
primordium but also at the junction of the midgut and the hindgut. This
region, which is called the small intestine
(Hoch and Pankratz, 1996
;
Snodgras, 1935
;
Strasburger, 1932
), is
characterised by extensive morphogenetic cell movements and thus displays many
similarities to the keyhole in the anterior region. Wingless-dependent gap
junctional communication may also be required in this area of the
gastrointestinal tract, although we have not observed gross morphological
changes in this region in kropf mutants.
Whether the transcription factor complex that transduces the Wingless
signal and which is composed of the ß-catenin homologue Armadillo and
Tcf/Lef DNA-binding proteins, directly interacts with the regulatory region of
innexin 2 to activate its transcription, is presently unknown.
However it is noteworthy that a consensus Tcf/Lef binding site is found 5 kb
upstream of the putative innexin 2 promotor. Armadillo is distributed
throughout the foregut, but is concentrated in specific areas of the
developing proventriculus (Pankratz and
Hoch, 1995). At early stages, it is strongly expressed near the
keyhole region and at later stages it becomes highly concentrated in areas
undergoing the most extensive cell movements. The proventriculus phenotype of
armadillo mutant embryos is similar to that of wingless null
embryos, strongly suggesting the requirement of armadillo during
proventriculus morphogenesis. In addition, innexin 2 transcription is
induced by Armadillo in tissue culture cells. It is thus possible that
Armadillo and Tcf/Lef directly regulate innexin 2 transcription.
Apical localisation of innexin 2 gene products
Our expression studies show that innexin 2 mRNA is localised to
the apical side of cells. mRNA expression studies with other members of the
innexin family indicate a similar apical localisation pattern (C. L.
and M. H., unpublished). It is also noteworthy that wingless mRNA is
localised at the apical sides of epithelial cells
(Baker, 1987). The apical
localisation has been shown to depend on discrete elements within the
wingless 3`UTR (Simmonds
et al., 2001
). It requires cytoplasmic dynein protein that
assembles the apical RNAs selectively into particles that are transported
apically along microtubules (Wilkie and
Davis, 2001
). Redistribution of the transcripts causes a dramatic
loss of Wingless signalling activity. Furthermore, the subcellular
localisation of WNT transcripts also occurs in other organisms, suggesting a
conserved function (Simmonds et al.,
2001
). Various other components of the Wingless signalling
pathway, such as the Wingless transmembrane receptor Frizzled, the
ß-catenin homologue Armadillo, Dishevelled and the Armadillo-modifying
complex are also localised apically in the cells (for reviews, see
Wodarz and Nusse, 1998
;
Peifer and Polakis, 2000
). As
we have shown that innexin 2 is a target of Wingless signalling in
the proventricular cells, it is likely that apical innexin transcript
localisation is an important functional feature required for
Wingless-dependent gap junctional communication in the keyhole cells.
Furthermore, these data suggest the existence of yet unknown factors (most
probably RNA-binding proteins) that are required for apical innexin 2
mRNA localisation in epithelial cells. It remains to be shown whether connexin
mRNAs are also localised and, if so, what functional role the localisation
plays. Electrophysiological experiments in the heterologous Xenopus
system have demonstrated that Innexin 2 forms heteromeric gap junctions with
Innexin 3, another Innexin family member
(Stebbings et al., 2000
).
Whether heteromerisation is also required for keyhole formation is not known
because innexin 3 mutants have not been identified, yet.
The WNT/Wingless pathway regulates the transcription of gap junction
multigene families
Our results suggest that although invertebrate innexin genes share no
sequence homology to the vertebrate connexin genes
(Phelan et al., 1998a;
Phelan and Starich, 2001
),
both multigene families might be regulated by an evolutionarily conserved
signalling pathway, the WNT/Wingless signalling cascade. In the developing
Xenopus embryo, it has been observed that Wnt1 expression leads to an
enhancement of gap junctional communication in ventral cells (Olson et al.,
1991; Olson and Moon, 1992; Krufka et al.,
1998
). Studies in the mouse have shown that ectopic expression of
Wnt1 in the limb mesenchyme results in an increase of connexin 43
transcription (Meyer et al.,
1997
). Recent tissue culture studies using rat PC12 cell lines and
cardiomyocytes have further provided molecular evidence that connexin
43 is a downstream target gene of WNT1 signalling
(van der Heyden et al., 1998
;
Ai et al., 2000
). Our studies
about innexin 2 regulation and the evidence from the
connexin regulation in vertebrates suggest that WNT/Wingless
signalling may be an evolutionarily conserved signalling pathway regulating
the expression of gap junction-encoding multigene families.
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Acknowledgments |
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