Department of Medical Genetics and Microbiology, Collaborative Program in Developmental Biology, University of Toronto, Toronto, ON, M5S 1A8, Canada
* Author for correspondence (e-mail: peter.roy{at}utoronto.ca)
Accepted 22 April 2005
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SUMMARY |
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Key words: Muscle arms, Membrane extension, Myosin, actin, ADF/cofilin, Tropomyosin, unc-54, lev-11, unc-60, Caenorhabditis elegans
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Introduction |
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Adult C. elegans have 95 body wall muscles (BWMs) that are
required for locomotion and movement of the head. These muscle cells are
arranged in four quadrants along the length of the animal
(Fig. 1). The two dorsal
quadrants flank the dorsal cord, while the two ventral quadrants flank the
ventral cord. Within each quadrant there are two longitudinal rows of muscle
cells, a proximal row close to the nerve cord, and a distal row further from
the cord that we refer to as distal BWMs. The BWMs of nematodes establish
contact with motor axons using specialized membrane extensions called muscle
arms (Stretton, 1976;
Sulston and Horvitz, 1977
;
Sulston et al., 1983
;
White et al., 1986
). Muscle
arms have a simple morphology consisting of a thin stalk that emanates from
the cell body and a bifurcated terminus that contacts the nerve cord. The 16
anteriormost BWMs in the head extend muscle arms exclusively to motor axons in
the nerve ring (Fig. 1).
Immediately posterior to the head muscles, 16 neck BWMs extend arms both to
the nerve ring and to the nearest nerve cord. The remaining 63 BWMs extend
muscle arms exclusively to motoneurons located in the nearest nerve cord. By
contrast to the muscle membrane extensions observed during neuromuscular
junction formation in Drosophila, rat and mouse, C. elegans
muscle arms are maintained throughout the life of the animal.
How muscle arms reach the nerve cord is not clear. However, two lines of
evidence suggest that a chemoattractant guides the migration of muscle arms to
the nerve cords. First, muscle arms extend to motor axons irrespective of
their physical position. For example, commissural motor axons in
unc-6/netrin or unc-5/RCM mutants do not complete their
ventral-to-dorsal migrations and instead migrate along the longitudinal axis
at sublateral positions. In these mutants, the dorsal BWM arms that normally
extend to the motoneurons of the dorsal cord instead extend to the errant
lateral motor axons (Hedgecock et al.,
1990). Second, muscle arms extend to locations of dense core
vesicle accumulation in the cell bodies of motoneurons in unc-104
mutants (Hall and Hedgecock,
1991
; Zhou et al.,
2001
). This observation suggests that the dense core vesicles
contain a muscle arm chemoattractant. unc-104 encodes a kinesin motor
protein that is required for anterograde transport of vesicles within axons
(Hall and Hedgecock, 1991
;
Zhou et al., 2001
). These two
lines of evidence suggest that muscle arm migration to the nerve cords may be
analogous to axon extension toward the source of a chemoattractive cue.
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Here we present a detailed characterization of muscle arm development and have found it to be stereotypical, suggesting a high degree of genetic regulation. We also show that perturbation of various components of the actin and myosin machinery, including actin, tropomyosin, ADF/cofilin and muscle myosin, impair muscle arm extension and disrupt arm morphology. Our work has resulted in new insights about the mechanics of membrane extension in muscle cells and establishes C. elegans muscle arms as a useful in vivo model for membrane extension.
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Materials and methods |
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To investigate larval muscle arm extension in a post-embryonic cell
division mutant we made lin-6(e1466) dpy-5(e61)/hT2[qIs48]
(Wang and Kimble, 2001)
animals transgenic for him-4p::Mb::YFP. lin-6(e1466) homozygotes are
easily recognized, as the mutation is linked to dpy-5(e61), which has
a distinct phenotype. Post-embryonic divisions occur in lin-6(e1466)
mutants, but most daughter cells eventually die
(Sulston and Horvitz, 1981
).
The molecular identity of lin-6 is not published.
Microscopy, temperature shift experiments and statistical analysis
For microscopy, worms were anaesthetized in 2-10 mmol/l Levamisole (Sigma)
in M9 solution (Lewis, 1995) and mounted on a 2% agarose pad. We used a Leica
DMRA2 HC microscope with standard Leica filter sets for GFP, YFP, CGFP and GFP
Red epi-fluorescence to take all pictures. A 20x dry objective was used
to take all pictures of adult worms, while a 63x oil objective was used
to take pictures of young larvae and GFP::actin-expressing muscles. All muscle
arm counts were done from photographs, which are available upon request. To
study populations of worms at defined developmental stages, worms were first
synchronized (Lewis, 1995) and then staged by the extent of germline and gonad
cell proliferation (Kimble and Hirsh,
1979).
The temperature-shift experiments were done by incubating worms at either 15 or 25°C for at least 24 hours. Synchronized L1s (Lewis, 1995) were deposited on plates with food and placed at the desired temperature. The developmental stage of the worms was monitored and pictures of the dorsal right muscle quadrants were taken when the animals reached adulthood.
We did not assume Gaussian distributions of the number of counted arms and therefore used a more stringent two-tailed Mann-Whitney rank sum test to assess the statistical significance of the observed differences.
RNAi
All RNAi experiments were done by feeding worms dsRNA-producing bacteria as
previously described (Timmons and Fire,
1998). The pPRRF215 unc-54(RNAi) construct was made by
isolating a 1700 bp PstI-BglII unc-54 genomic
fragment that contains exons 4, 5 and the first 286 bp of exon 6 from the
pPD5.41 unc-54 genomic clone (a gift from Dr A. Fire) and sub-cloning
it into the RNAi feeding vector pPD129.36
(Timmons and Fire, 1998
) cut
with PstI and BglII. pPRRF215 and the negative
control (empty pPD129.36) vector were transformed into HT115 bacteria
(Timmons et al., 2001
) using
standard protocols.
Muscle arm width analysis
The width of muscle arms from dorsal right BWM number 11 was measured for
ten young adult animals of each genotype using Openlab 3.1.5 software
(Improvision Inc.). Arm width was measured at the midpoint of each arm,
halfway between the body of the muscle and the nerve cord. Because the length
of worms, and therefore the length or size of BWMs, can vary substantially
between adults of different ages and of different genetic backgrounds, the arm
widths were normalized to the total muscle length along the anterior-posterior
axis to control for differences in animal size.
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Results |
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Expression of Mb::YFP from him-4p in first larval-stage (L1)
larvae was limited to four distal BMWs posterior to the neck muscles in each
quadrant (Fig. 2A). As
development continued, two more distal BWMs posterior to the initial four in
each quadrant also expressed Mb::YFP, albeit at lower levels
(Fig. 1D,E). We were able to
assign unambiguous identities to the 16 BWMs that expressed Mb::YFP brightly
from him-4p because of the largely invariant cell lineage and the
stereotypical pattern of BWM shapes and arrangements
(Sulston and Horvitz, 1977;
Sulston et al., 1983
). For
example, the dorsal right BWM Cppapap is the most anterior muscle that is
always overlapped by neighbouring BWMs on either side and can therefore serve
as a useful position marker (Fig.
1F). There are 14 BWMs born post-embryonically from the
M-mesoblast cell that intercalate into the existing quadrants at variable
positions (Sulston and Horvitz,
1977
). The general integration space is posterior to the gonad
primordium, which is located just posterior to the four brightly fluorescing
BWMs. The identities we assigned to the posterior muscles that fluoresced
lightly are therefore based on position only and not lineage. For convenience
we have renamed the BWMs with numbers according to their position along the
anterior-posterior axis in each quadrant. For example, Cppapap is referred to
as dorsal right muscle 15 (Fig.
1F,G).
To control for the possibility that the Mb::YFP muscle arm reporter may interfere with muscle arm outgrowth, migration or morphology, we examined BWMs expressing DsRed2 driven from the muscle-specific C26G2.1 promoter (P.J.R. and S. Quaggin, unpublished). There were no differences in arm morphology or the number of arms observed with the two different reporters (not shown and Table 2, line 35). We conclude that Mb::YFP driven from him-4p does not interfere with muscle arm number or morphology and is therefore a useful tool to study muscle membrane extension.
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The UNC-104 kinesin motor protein is thought to transport the putative
muscle arm chemoattractant along motor axons
(Hall and Hedgecock, 1991). We
therefore counted the number of muscle arms extended in the background of
three unc-104 loss-of-function mutations, including the null mutation
rh142 (Hall and Hedgecock,
1991
). In this and all further mutant analysis we considered the
muscle arms only from the dorsal right quadrant BWMs, as all quadrants show
similar defects and muscle arms are most clearly visible in this quadrant. The
unc-104 mutants displayed significantly fewer muscle arms than did
wild-type adult animals (P<0.001)
(Table 2, lines 31-33;
Fig. 3). Intriguingly,
unc-104 mutant adults extended similar numbers of muscle arms to
wild-type hatchlings. This suggests that UNC-104 is required for larval muscle
arm outgrowth, but is dispensable for embryonic muscle arm development.
|
Actin loss-of-function mutations have no obvious phenotype, probably
because of genetic redundancy. We therefore used an actin gain-of-function
mutation (st15) to examine the consequences of actin perturbation on
muscle arm development. The act-1,2,3(st15) allele is a mutation
within the actin gene cluster act-1,2,3, although it is unknown which
of the three actin genes is mutated
(Landel et al., 1984;
Waterston et al., 1984
). We
observed three muscle arm phenotypes in the act-1,2,3(st15)
background. First, act-1,2,3(st15) adults had significantly fewer
arms than controls, but more than L1 hatchlings
(Table 2, line 10). Second, 91%
of the mutant BWMs extended membrane in the general direction of the nearest
nerve cord but did not make contact with it, compared with 10% of controls
(Fig. 3,
Table 3). We refer to this
phenotype as errant membrane projections. Third, there was a dramatic
arborization of muscle arm termini at the nerve cords
(Fig. 3,
Table 3). In addition to these
arm phenotypes, 73% of act-1,2,3(st15) BWMs had flowing lateral ends
(Fig. 3B), which differed
significantly in morphology from the pointed lateral ends of wild-type
controls (Table 3). We also
examined the distribution of actin in the st15 mutant background
using the GFP::ACT-1 reporter. We found that muscle arm termini in
act-1,2,3(st15) mutants were enriched with actin compared with
controls (Fig. 4). Moreover, we
were unable to resolve individual actin filaments or actin filament bundles
within the arm termini. These results demonstrate a crucial role for actin
regulation in muscle arm development.
|
|
We observed three muscle arm phenotypes in unc-60B(su158) animals. First, unc-60B(su158) animals have as few muscle arms as wild-type L1 hatchlings (Fig. 3) (Table 2, line 11). This suggests that larval muscle arm extension is abrogated in animals lacking UNC-60B. Second, 70% of unc-60B(su158) BWMs have errant membrane projections (Fig. 3) (Table 3). Third, 29% of unc-60B(su158) BWMs have arborized arm termini, compared to 2% of controls (Table 3). In addition, 100% of the BWMs in an unc-60B(su158) background have flowing lateral ends, compared to none in controls (Fig. 3) (Table 3). The F-actin bundles in the muscle arms of unc-60B(su158) mutants appear disorganized in the shaft and there is an accumulation of actin fibres within the muscle arm termini, similar to act-1,2,3(st15) mutants (Fig. 4). These results suggest an important role for UNC-60B-mediated actin regulation in muscle arm extension.
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Tropomyosin is required for both muscle arm extension and morphology
ADF/cofilin-dependent actin disassembly is antagonized by tropomyosin,
which structurally reinforces actin filaments
(Bernstein and Bamburg, 1982;
Blanchoin et al., 2000
;
DesMarais et al., 2002
;
Ono and Ono, 2002
). We
investigated whether C. elegans tropomyosin, called LEV-11 or TMY-1,
might also be required for muscle arm extension. We examined muscle arms in
two different lev-11(RNAi) backgrounds. lev-11(TM1-RNAi)
targets the two lev-11 isoforms that are thought to be
muscle-specific, while lev-11(TM2-RNAi) targets isoforms that are
expressed in BWMs, the pharynx and the intestine
(Kagawa et al., 1995
;
Ono and Ono, 2002
). Both
lev-11(TM1-RNAi) and lev-11(TM2-RNAi) resulted in a
significant reduction in the number of muscle arms in adults
(Table 2, lines 16 and 17). We
therefore restricted further lev-11(RNAi) analysis to the TM1
isoforms. As the number of muscle arms in lev-11(RNAi)-treated
animals is wild-type hatchlings (P>0.05), we conclude that
tropomyosin is essential for larval arm extension.
Tropomyosin stabilizes actin filaments
(DesMarais et al., 2002;
Ono and Ono, 2002
). We
therefore tested whether lev-11(RNAi) could suppress the membrane
extension defects observed in unc-60(su158) and
act-1,2,3(st15) mutants, which probably result from excessive actin
filament extension or stability. We observed that unc-60(su158);
lev-11(RNAi) worms had fewer BWMs with errant membrane projections (19%
vs 70%), flowing lateral muscle ends (83% vs 100%), and fewer muscle arms with
arborized arm termini (17% vs 29%) than unc-60(su158) mutants alone
(Table 3).
lev-11(RNAi) similarly reduced the membrane-associated defects
observed in an act-1,2,3(st15) background
(Table 3). These results show
that unc-60 and lev-11 act antagonistically in BWMs and are
consistent with previous in vitro studies
(Ono and Ono, 2002
).
|
Muscle myosin is required for muscle arm extension and morphology
Many of the mutants and RNAi-treated animals that we have examined are not
only predicted to have disrupted actin dynamics, but are also severely
uncoordinated. To test if muscle arm development is dependent on locomotion,
we examined muscle arms in the background of two mutations that result in
severely uncoordinated movement but were not known to disrupt actin
regulation. First, we examined muscle arms in animals mutant for
egl-30, which encodes an -subunit of heterotrimeric G protein
(Brundage et al., 1996
).
egl-30(n715) mutants were paralyzed, but had no muscle arm
phenotypes, demonstrating that paralysis alone does not disrupt muscle arm
development (Table 2, line 34;
Fig. 6). We then examined
unc-54, which encodes muscle myosin heavy chain B (MHC-B)
(MacLeod et al., 1977
).
Surprisingly, unc-54 loss of function resulted in significantly fewer
arms and greater arm widths than wild-type controls
(Table 2, line 20;
Fig. 6).
MHC-B is expressed in all muscles in C. elegans except for the
pharynx (Ardizzi and Epstein,
1987; Okkema et al.,
1993
) and is one of three components of thick filaments in BWMs,
together with MHC-A, encoded by myo-3, and paramyosin, which is
encoded by unc-15 (Kagawa et al.,
1989
; Miller et al.,
1986
; Schachat et al.,
1977
; Waterston et al.,
1977
). Because MHC-B is an integral component of thick filaments,
we investigated if disruption of this structure is likely to be the primary
cause of the muscle arm defects observed in unc-54 nulls. First, we
examined muscle arms in unc-15 mutants, which contain MHC-B
aggregates in the BWMs and do not form thick filaments
(Waterston et al., 1977
).
Table 2 and
Fig. 6 show that
unc-15 loss-of-function mutants exhibited only a small decrease in
the number of muscle arms and no increase in muscle arm width. This suggests
that wild-type thick filaments are dispensable for muscle arm development.
Next, we examined muscle arms in reduction-of-function backgrounds of
myo-3. As the two available myo-3 mutants are lethal, we
first examined myo-3 reduction-of-function by RNAi and found no
muscle arm defects in either a wild-type background or the RNAi-sensitive
background, rrf-3(pk1426) (data not shown). We then examined
myo-3(st386) animals that were mosaic for a
myo-3p::MHC-A::GFP rescuing extra-chromosomal array stEx30
(Campagnola et al., 2002
). The
number of muscle arms extended by muscles expressing MHC-A::GFP was not
significantly different from muscles without MHC-A (P>0.01;
Fig. 7). In addition, there is
no significant difference in the arm widths of muscles with or without MHC-A
(P>0.01). Together, these results strongly suggest that the muscle
arm defects observed in unc-54 mutants are not a secondary
consequence of disrupted thick filaments. We conclude that muscle myosin heavy
chain B is specifically required for muscle arm development.
|
unc-54(e190) mutants resembled lev-11(RNAi)-treated animals: the number of muscle arms was significantly reduced, the width of the remaining arms was significantly increased, the arm termini were infrequently arborized, and the BWMs did not display lateral flowing ends or membrane extension defects (Table 3). If MHC-B and LEV-11 regulate the same process, then lev-11(RNAi)-treated unc-54(e190) nulls should not have fewer muscle arms than lev-11(RNAi)-treated worms alone. Consistent with this hypothesis we found that unc-54(e190); lev-11(RNAi) animals had equivalent numbers of muscle arms to lev-11(RNAi) animals (Table 2, line 28). Furthermore, muscle arm widths of unc-54(e190); lev-11(RNAi) animals were not significantly different from those of lev-11(RNAi) animals and were not additive (P>0.05). This suggests that MHC-B and LEV-11 function together to regulate muscle arm extension and morphology.
Strikingly, unc-54(RNAi) enhanced the arm termini arborization
phenotype in both act-1,2,3(st15) and unc-60(su158) mutants
(Fig. 5,
Table 3). This enhancement was
both quantitative and qualitative, as the arborization was continuous from
muscle to muscle in almost all act-1,2,3(st15); unc-54(RNAi) animals
and in some unc-60(su158); unc-54(RNAi) animals. It is
intriguing that these animals also had defasciculated nerve cords. Because
unc-54 and unc-60B are specifically expressed in muscle and
not neurons (Ardizzi and Epstein,
1987; Okkema et al.,
1993
; Ono et al.,
1999
), our results suggest that the arborization of muscle arms
can lead to nerve cord defasciculation. In addition, the enhancement of arm
arborization by unc-54(RNAi) suggests that MHC-B normally restricts
actin-based membrane extension in the muscle arm termini.
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Discussion |
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A `two-phase' model of muscle arm development
Adults that have null mutations in unc-60B/ADF/cofilin or
unc-54/MHC-B, or have been treated with
lev-11/tropomyosin(RNAi), have similar numbers of arms to
wild-type hatchlings. It is unlikely that these genes have redundant functions
in arm extension because various double mutant combinations fail to further
reduce the number of muscle arms. Rather, we propose a `two-phase' model of
muscle arm development in which larval muscle arms develop by a fundamentally
different mechanism from embryonic muscle arms
(Fig. 9). Previous studies have
shown that myoblast progenitors of the head and neck BWMs are born juxtaposed
to the nerve ring. As embryonic development proceeds, these myoblasts move
away from the nerve ring and leave muscle arms behind (C. R. Norris, I. A.
Bazykina, E. M. Hedgecock, D. H. Hall, personal communication). As none of the
cytoskeletal mutants we examined had fewer arms than hatchlings, we infer that
all BWMs passively leave membrane connections behind as they move away from
the nerve cords during embryogenesis (Phase I). We propose that BWMs actively
extend muscle arms only during larval development (Phase II). Our model is
supported by two additional observations. First, unc-54/MHC-B is
required only for the extension of larval muscle arms, as unc-54
mutant hatchlings have the same number of arms as wild-type hatchling
controls. Second, the number of muscle arms in unc-104 mutant adults
is similar to wild-type hatchlings. Because UNC-104 is required to transport
vesicles that probably carry the putative chemotropic cue, the remaining
muscle arms in the unc-104 mutants must not rely this cue for their
development.
A novel role for muscle myosin in membrane extension
We have discovered unexpected roles for myosin heavy chain B
(unc-54) in both phases of muscle arm development. First, MHC-B is
required to regulate embryonic muscle arm morphology, as unc-54
mutations result in a wide arm phenotype. The same phenotype is observed in
lev-11(RNAi)-treated animals. Furthermore,
lev-11(RNAi)-treated unc-54 nulls do not have wider arms
than either single gene disruption alone. These results suggest that MHC-B and
tropomyosin act together to regulate muscle arm morphology and are consistent
with the known role of tropomyosin in mediating myosin-actin interactions in
both muscle and non-muscle cells (Bryce et
al., 2003; Gordon et al.,
2000
).
In our two-phase model of arm development, embryonic muscle arms are the
trailing edge remnants of myoblast movement away from the motor axons.
Intriguingly, inhibition of non-muscle myosin II activity in migratory
neutrophils results in a failure to retract the trailing edge, which
consequently expands in size and accumulates actin filaments
(Eddy et al., 2000). It has
been proposed that non-muscle myosin II generates tension within the
cytoskeleton of the trailing edge and promotes dissociation of adhesion
complexes from the substrate, allowing forward cell translocation
(Eddy et al., 2000
). We
similarly propose that MHC-B, which is a muscle myosin II family member
(Hodge and Cope, 2000
;
Sellers, 2000
), also generates
actin-filament tension within the developing embryonic muscle arms to restrict
arm width. Our model is supported by the observations that disruption of
unc-54 results in the expansion of embryonic muscle arms, the
accumulation of actin within the arm terminus, and the dramatic enhancement of
the actin-based arborization of termini in unc-60B and
act-1,2,3 mutants.
MHC-B is also required for the active extension of muscle arms during the
second phase of muscle arm development. The number of muscle arms in
unc-54 null hatchlings is identical to wild-type hatchlings.
Similarly, adult animals in which unc-54 was compromised during early
larval development using a temperature-sensitive allele have a similar number
of muscle arms as wild-type hatchlings. These results demonstrate that MHC-B
is essential for larval muscle arm extension. This is the first evidence that
the muscle myosin II MHC-B can play an active role in membrane extension.
However, non-muscle myosin II is well known to be required for membrane
extension in many cell types (Diefenbach et
al., 2002; Wylie et al.,
1998
; Lo et al.,
2004
; Pollard and Borisy,
2003
). We speculate that unc-54 mutants fail to extend
larval arms because MHC-B is needed to generate the actin-based tension
required for membrane extension at the leading edge as previously described in
other systems (Diefenbach et al.,
2002
). Our observation that unc-54(RNAi) and
lev-11(RNAi)-treatments can suppress the lateral flowing BWM ends and
membrane extension phenotypes seen in the actin gain-of-function mutant
further supports this model.
The regulation of actin dynamics during larval muscle arm extension
UNC-60B/ADF/cofilin has two important biochemical activities required for
the proper assembly and maintenance of the C. elegans sarcomere:
F-actin depolymerization and severing (Ono
et al., 1999). Although all the unc-60B alleles that we
examined disrupt sarcomere development, only those alleles that inhibit the
depolymerizing activity of UNC-60B have larval muscle arm extension defects.
This suggests that it is the F-actin depolymerizing activity of
UNC-60B/ADF/cofilin, and not the severing activity, that is crucial to
membrane extension in muscle. These observations contradict a previous report
that only the severing activity of ADF/cofilin is required for membrane
extension in transgenic chick neurons
(Endo et al., 2003
). Although
both studies rely on previous biochemical analysis of mutant ADF/cofilin
activity, it is possible that the mutations behave differently in vivo, or
have neomorphic activities that were not considered. Alternatively, chick
cofilin and C. elegans UNC-60B may have either divergent properties
or are regulated differently in neurons or muscle cells. In any case, our
results suggest that enzymatic modulation of the actin cytoskeleton is crucial
for muscle arm extension.
In summary, we have demonstrated that muscle arms are a useful model system to study the mechanics of membrane extension because they are readily observed in living animals and are amenable to genetic disruption. Indeed, this work has led to novel insights into the functions of conserved cytoskeletal components and regulators. We anticipate that the study of muscle arms in C. elegans will be valuable in uncovering new genes required for membrane extension and guided migration.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/13/3079/DC1
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ACKNOWLEDGMENTS |
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