1 Program in Neuroscience and Department of Physiology, University of
California, San Francisco, 513 Parnassus Avenue Box 0444, San Francisco, CA
94143, USA
2 Department of Anatomy and Neurobiology, Washington University School of
Medicine, 660 South Euclid, St Louis, MO 63110, USA
Author for correspondence (e-mail:
hbaier{at}itsa.ucsf.edu)
Accepted 28 November 2003
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SUMMARY |
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Key words: Retina, Neural differentiation, Axon guidance, Target selection, Zebrafish
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Introduction |
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The highly laminated structure of the retina makes it an ideal model system
for studying the mechanisms of synaptic layer formation. In the mature inner
plexiform layer (IPL; Fig. 1A),
connections between retinal ganglion cells (GCs) and their presynaptic
partners, the amacrine cells (ACs) and bipolar cells (BCs), are broadly
segregated into ON or OFF sublayers, each representing circuits that are
either depolarized (ON) or hyperpolarized (OFF) by increased illumination
(Famiglietti and Kolb, 1976).
The ON and OFF sublayers are further divided into functionally specialized
sublaminae (e.g. Roska and Werblin,
2001
; Wässle and Boycott,
1991
). The IPL and its sublaminae extend continuously across the
retina, a feature that is important for lateral processing of visual
information. Elucidating the mechanisms underpinning IPL formation and
sublamination will provide important insights into how neural circuits become
precisely wired during development.
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Although elegant in design, these experiments do not unequivocally rule out
a role for GCs in IPL organization, because GCs were present and had
differentiated prior to ablation. Furthermore, some ACs are already stratified
at birth in rodents (Reese et al.,
2001; Stacy and Wong,
2003
), prior to the manipulations that eliminated GCs.
We thus investigated the role of GCs in organizing lamination of the IPL
using a zebrafish mutant in which GCs are never present. The lakritz
(lak) gene encodes Atonal homologue 5 (Ath5), a
basic-helix-loop-helix-transcription factor required specifically for the
genesis of GCs (Kay et al.,
2001; Brown et al.,
2001
; Wang et al.,
2001
). In lak mutants, the first wave of retinal
neurogenesis, in which GCs are normally made, fails to occur. As a result,
progenitors are prevented from adopting the GC fate
(Kay et al., 2001
). However,
ACs and all other cell types differentiate on time, and an IPL is present
(Kay et al., 2001
). The
specificity of this genetic lesion allowed us to analyze the behavior of ACs
in the absence of GCs. For this purpose, we generated transgenic zebrafish in
which stable subpopulations of ACs express green fluorescent protein (GFP).
GFP expression in these lines marks ACs earlier in their development than most
other known markers (Pow et al.,
1994
), allowing us to visualize the dynamic behavior of
GFP-labeled AC neurites in vivo from the earliest stages of neurite outgrowth.
Because the zebrafish retina develops rapidly, we were able to continuously
monitor IPL development from its formation until segregation into ON and OFF
sublaminae, providing the first in vivo view of how functionally distinct
synaptic layers arise in a CNS structure during development. Our observations
reveal that whereas elimination of GCs causes specific temporal and spatial
perturbations in IPL formation, amacrine cells can assume a central role in
the formation of synaptic laminae in the retina.
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Materials and methods |
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The linearized plasmid (Fig.
1B) was injected into zebrafish embryos at the one-cell stage,
following standard procedures (Stuart et
al., 1988; Higashijima et al.,
1997
). The injected embryos were selected for transient GFP
expression one day later and raised to adulthood. We recovered nine founders
from 70 injected fish (13%), a rate within the range of those previously
reported for this technology. Founder fish were crossed to non-transgenic
fish, and GFP-expressing progeny were raised to generate a stable line. To
visualize IPL formation, the experiments in this study were carried out on
three lines: Pax6-DF4:mGFPs220,
Pax6-DF4:mGFPs243, and
Pax6-DF4:mGFPs244. We will refer to these lines as 220,
243 and 244 throughout this paper. We also generated several lines with a
similar construct, which was lacking the membrane-targeting signal, but was
otherwise identical to the one shown in
Fig. 1B. Six of 26 injected
fish (23%) transmitted the transgene to the next generation.
Identification of lak mutants
Homozygous lak mutant embryos (lakth241/th241)
(Kelsh et al., 1996) were
identified at 2 days post-fertilization (dpf) in live imaging experiments by
the abnormal laminar position of GFP+ ACs. This method was
validated by RFLP genotyping for the lakth241 allele
(Kay et al., 2001
). Older
larvae were scored as lak mutants based on their visual background
adaptation defect, which results in dark pigmentation
(Kelsh et al., 1996
), or, for
some experiments, by immunostaining for GCs
(Kay et al., 2001
).
Immunocytochemistry
ChAT
Adult 220 transgenic fish were euthanized using Tricaine (0.02% w/v in
0.3xDanieau's solution). Following decapitation, their eyes were removed
and retinas dissected out and fixed in 4% paraformaldehyde (PFA) in 0.1 M
phosphate-buffered saline (PBS), pH 7.4, for 2.5 hours. Vibratome sections (50
µm) were cut and incubated in anti-ChAT (AB144P, Chemicon) diluted 1:100 in
PBS containing 5% normal donkey serum (NDS) and 0.5% Triton X-100, for 36
hours with gentle agitation. After several rinses in PBS, sections were
blocked with 5% NDS in PBS for 1 hour and then incubated in secondary
antibody, Alexa 568 donkey anti-goat (Molecular Probes) at 1:1000 in 5% NDS in
PBS for 2.5 hours with gentle agitation. After several rinses in PBS, sections
were cover-slipped in Gelmount.
PKC
The progeny of a 220/+; lakth241/+ incross were fixed
in 4% PFA/PBS at 5 dpf, cryosectioned (12 µm), and immunostained for PKC as
described (Kay et al., 2001).
Single confocal scans were used to detect PKC immunofluorescence and native
GFP fluorescence simultaneously.
GFP
Whole-mount embryos or cryosections were stained as described
(Kay et al., 2001) using
rabbit anti-GFP (Molecular Probes A-11122; 1:4000 dilution). To permeabilize
3.5 dpf embryos, they were incubated with 0.1% collagenase in PBS for 90
minutes prior to antibody staining.
Live imaging
Embryos/larvae were maintained in embryo medium (0.3xDanieau's
solution with 100 units/ml penicillin and 100 µg/ml streptomycin) at
28.5°C prior to imaging. At 15 hours post-fertilization (hpf),
1-phenyl-2-thiourea (PTU) was added to embryo medium at a final concentration
of 0.003% (w/v) in order to block pigmentation in the eye. Prior to imaging,
chorions were manually removed and the larvae transferred to a solution of
embryo medium containing 0.003% PTU and 0.02% (w/v) Tricaine. Larvae were then
embedded in a petri dish (Falcon # 35-3037) in 0.5% low melting point agarose
dissolved in embryo medium containing PTU and Tricaine. Dishes were placed in
a temperature controlled stage (Cell Microtemp Systems) and recordings were
performed at 28-30°C. Typically up to ten fish were aligned in series to
enable imaging several fish during each recording session. Images were
acquired and presented primarily from the peripheral retina because the lens
distorted images of the central-most part of the retina.
Confocal image stacks were acquired using either the BioRad 1024M or Olympus FV500. Long working distance water objectives, including 20x Nikon (NA 0.5), 40x (NA 0.8) and 60x (NA 0.9) Olympus objectives, were used. To reconstruct cells and their processes, a series of optical planes were collected in the z dimension (z-stack), and collapsed into a single image (maximum intensity or z-projection) or rendered in three dimensions to provide views of the image stack at different angles. The step size for each z-stack was chosen upon calculation of the theoretical z-resolution of the objective used (typically 0.5-1.0 µm). Time-lapse imaging was carried out by collecting z-stacks encompassing the same cells within a selected region per retina at various intervals (typically once every hour or more, and for total recording periods up to around 40 hours). Image analysis was performed offline, using Metamorph (Universal Imaging) to generate image alignments, orthogonal rotations and movies of the z-stacks.
Generation and analysis of chimeras
Chimeric embryos were generated by standard methods
(Ho and Kane, 1990). The
progeny of a 220/220; lakth241/+ incross were used as
donors, and non-transgenic lakth241/+ incross progeny were
used as hosts. At the 1000-cell stage, 10-50 cells were removed from a donor
and transplanted to the animal pole of the host blastula. Donors were
genotyped by RFLP (Kay et al.,
2001
). Chimeras were treated with PTU, fixed at 3.5 dpf (after AC
sublamination) and immunostained in whole mount with anti-GFP. Individual ACs
were imaged on a confocal microscope (BioRad 1024) and reconstructed (see
above). For some experiments, chimeras were allowed to survive until 7 dpf,
and then sectioned and immunostained with anti-GFP. Only sections taken
through the center of the retina (as determined by maximal lens diameter) were
used to score sublamination of GFP+ AC neurites, thereby preventing
section angle from confounding the analysis. Images were collected using
single confocal scans.
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Results |
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The Pax6 homeodomain transcription factor is expressed in all retinal
neuroblasts (Marquardt et al.,
2001), a feature that is recapitulated in our lines
(Fig. 2A). Later in
development, endogenous Pax6 becomes restricted to a subset of ACs in goldfish
(Hitchcock et al., 1996
),
zebrafish (Masai et al., 2003
)
and mouse (Marquardt et al.,
2001
). A similar restriction of DF4-driven GFP to ACs is also
observed in our lines. However, Pax6-immunoreactive ACs are much more numerous
than the GFP+ ACs (Masai et
al., 2003
). Thus, our reporter lines, although providing a useful
in vivo marker for ACs, do not fully reproduce the endogenous Pax6 expression
pattern.
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Time-lapse imaging revealed that GFP+ AC neurites are
progressively added to the forming IPL in a `clockface' progression
(Fig. 2B; see Movie 1 at
http://dev.biologists.org/
supplemental). The nascent IPL recruits neurites from adjacent regions in
order to propagate laterally. The progression of IPL innervation trails the
anterior-to-posterior wave of AC neurogenesis
(Hu and Easter, 1999) by
10 hours. The GFP+ plexus is initially unstratified;
sublamination of GFP-labeled neurites starts in the developmentally more
advanced (nasoventral) part of the retina at around 65 hpf and spreads
dorsally, again in a clockface progression
(Fig. 2C; see Movie 2 at
http://dev.biologists.org/supplemental).
Sublamination commences at approximately 20 hours after an IPL first becomes
visible and is complete throughout the retina by about 90 hpf, an age that
roughly coincides with onset of behavioral responses to moving stimuli
(Easter and Nicola, 1996
).
IPL formation is delayed in lak mutants
We next used lak mutants to ask how the IPL develops in the
absence of GCs, the major postsynaptic target of the ACs
(Fig. 3; see Movies 3A,B). Our
time-lapse recordings revealed that the earliest steps of IPL formation were
severely affected in lak mutants. First, we observed a marked delay
in the formation of the IPL. As illustrated in
Fig. 3, an IPL is present in
the wild-type inner retina at 50 hpf but it is not apparent until 55-57 hpf in
the lak retinas. Quantitative analysis confirms this observation: Of
45 siblings concurrently imaged from 48 to 85 hpf, an IPL had appeared in 89%
of wild-type retinas (32 of 36) at the earliest time point (48-52 hpf). By
contrast, only 11% (1/9) of lak mutants in this clutch had developed
an IPL by 54 hpf. In a separate experiment, we sorted embryos according to the
presence or absence of an IPL at 48-50 hpf. Although 97% of wild-type siblings
(33/34) possessed an IPL at this age, only 22% (2/9) of mutants had an IPL,
thus supporting our conclusion that IPL appearance is delayed in the absence
of GCs. Nevertheless, all lak mutants eventually did develop an IPL,
indicating that GCs are not essential for its formation.
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The disorganization of the nascent lak mutant IPL suggests that GCs impart order on AC neurites as they begin to project to the IPL. However, some of the errors we observed in lak mutants appear to result primarily from exuberant growth of ACs towards each other. In particular, the clumping of ACs and their neurites outside the IPL (Fig. 4D-G) suggested that ACs might have a tendency to attract each other. To look more directly for evidence of such cellular behavior, we made time-lapse recordings of line 243 in the lak background. A sample recording shows individual cells that project neurites not towards landmarks such as the ILM or the IPL, but rather towards the neurites of other ACs (see Movie 5 at http://dev.biologists.org/supplemental). In this movie, neurites from two isolated ACs are seen approaching each other, making putative contact and forming a stable nexus of processes. Notably, each of the cells also appears to make contact with other nearby AC neurites, indicating that agglomeration errors may originate from AC-AC attraction. The lak mutant phenotype, therefore, reveals the existence of both GC-AC interactions and AC-AC interactions during formation of the IPL.
IPL sublamination occurs in the lak mutant, but is locally perturbed
We next turned our attention from IPL formation to sublamination, and asked
whether this process is affected by the absence of GCs. We found that
sublamination occurs late and is imperfect in lak mutants. By 82 hpf,
the GFP plexus of wild-type retina generally shows sublamination, but it
rarely does in lak mutants (Fig.
3; also see Fig. S3A,B at
http://dev.biologists.org/supplemental).
At 4 dpf, however, two distinct sublaminae have formed in mutants, similar to
wild type (Fig. 5). With age (7
dpf; Fig. 5), these sublaminae
were maintained, and the jagged course of the IPL appears to further
straighten. However, unlike wild-type animals where ON and OFF sublaminae are
parallel and continuous across the retina, sublamination is locally perturbed
in lak mutants. One or both of the sublaminae may be absent (often
replaced by a diffuse distribution of GFP+ processes throughout the
IPL), or an ectopic sublamina or non-laminar agglomerations may appear locally
(Fig. 5). Multiple errors were
always seen throughout the retina in every 5-7 dpf mutant eye examined
(n>40). Some of these non-laminar clumps resemble structures seen
in the nascent IPL of lak mutants
(Fig. 4), suggesting that these
errors occur early in development and persist to mature stages.
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The effect of the lak mutation on AC sublamination is cell-nonautonomous
Is the absence of GCs sufficient to explain the lak IPL phenotype?
The lak/ath5 gene is transiently expressed in most or all
retinal progenitors, including those that generate the ACs
(Masai et al., 2000) (J.N.K.
and H.B., unpublished). It is therefore important to investigate whether the
AC IPL phenotype is really due to absence of GCs, or whether the lak
mutation affects ACs directly. To address this issue, we created
lak/wild-type chimeras by transplanting small groups of cells from
220 carriers into non-transgenic embryos
(Ho and Kane, 1990
). We then
fixed the chimeras at 4 dpf and inspected their retinas in whole mount for the
presence of single GFP+ cells
(Fig. 7A-F). We found that all
wild-type ACs transplanted into a wild-type retina were monostratified, either
in the presumed ON or in the presumed OFF layer (n=28 cells
from 12 eyes; Fig. 7A,B,G).
When lak ACs were transplanted into wild-type retina, their
morphologies were indistinguishable from wild type (n=8 cells from 4
eyes; all monostratified; Fig.
7C). However, wild-type ACs transplanted into a lak
mutant retina occasionally (2/7 cells) had diffuse or bistratified processes
(n=7 cells from two eyes; Fig.
7D). The distribution of projection errors was similar to that
seen when lak ACs were transplanted into a lak host
(n=7 cells from two eyes; 2/7 diffuse;
Fig. 7E,F). These results
suggest that the projection phenotype of an AC is determined not by its own
lak genotype, but by that of the surrounding cells.
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Discussion |
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The zebrafish lak/ath5 mutant provides a unique experimental
situation in which the major postsynaptic cells of the inner retina, the GCs,
are never present. Loss of ath5 function causes a cell-fate switch
that blocks GC formation and increases the number of BCs
(Kay et al., 2001). However,
increased BC density is unlikely to explain the AC phenotypes we report here,
because these phenotypes can be observed even before BCs begin innervating the
IPL (Schmitt and Dowling,
1999
). The number of Pax6-DF4:GFP+ ACs is not
increased in lak mutants (Kay et
al., 2001
). Our cell transplantation experiments further convince
us that the lak mutant can be used as a specific tool to study the
effects of GC ablation on IPL development.
Time-lapse imaging of GFP-labeled AC neurites revealed the dynamics of IPL formation and ON/OFF sublamination in wild-type and mutant retinas. These studies demonstrate a previously unrecognized role for GCs in positioning and orienting AC neurites during their initial outgrowth towards the IPL. Our studies also highlight, however, the importance of ACs in IPL assembly: ACs can form the IPL on their own, and they appear to provide sublaminar targeting cues to their synaptic partners.
Migration and somal positioning of ACs are influenced by GCs
GCs are well placed to guide the migration of newborn ACs because they are
the first cell type to differentiate in the vertebrate retina. In wild type,
the early IPL invariably forms at the interface of GCL and the AC layer. In
lak mutants, IPL formation begins in an ectopic location, next to the
ILM. However, as ACs migrate towards the inner retina and take up positions
adjacent to the ILM, the IPL is repositioned towards the outer retina (see
Fig. 8E,F for a model). These
observations raise the possibility that GCs normally restrict the majority of
ACs from migrating into the GCL.
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Second, the disorganization of the AC projection in the lak mutant suggests that signals from GCs are important for AC neurite guidance. This phenotype is not caused by the absence of Ath5 in ACs, as shown by our transplantation experiments. One possibility is that the GC-derived signals act at a distance to orient AC growth towards the nascent IPL (Fig. 8D); another possibility is that contact between AC and GC neuritic processes stabilizes appropriately directed neurites while destabilizing misguided ones.
The severe IPL phenotype seen in lak mutants contrasts with the
relatively mild effects of GC ablation by optic nerve transection
(Günhan-Agar et al.,
2000; Williams et al.,
2001
). In the latter studies, GCs form but are experimentally
killed in newborn rats or ferrets. We suggest that the different outcomes of
these experiments demonstrate that even transient presence of GCs can pattern
the IPL.
ACs can form the IPL alone
GC-derived cues are not the only signals capable of guiding AC neurites,
given that many ACs in lak mutants either project normally to the IPL
or correct their early errors It is unlikely that BC terminals provide AC
guidance signals, as they have yet to innervate the IPL during the time when
ACs are doing so (Schmitt and Dowling,
1999) (J.N.K., A.M. and H.B., unpublished). Similarly, Müller
glia differentiate too late to guide the initial projection of ACs
(Peterson et al., 2001
). As
the lak mutant IPL consists predominantly of ACs at these early time
points, ACs do not seem to require interactions with other IPL constituents in
order to form an IPL-like plexus.
How do AC neurites form an IPL in lak mutants? It is possible that interactions with neuroepithelial cells might guide AC projections. However, our observations suggest another possibility, that AC-AC attraction may be sufficient for IPL formation. Many of the errors made by GFP+ ACs in lak mutants are most easily explained if we postulate that ACs can attract each other: The tendency of AC somata and neurites to clump, and the observation of newly differentiating adjacent ACs extending neurites directly towards each other, rather than towards the IPL, are particularly suggestive of attractive behavior. Our imaging study cannot provide definitive proof of AC-AC attraction, but we hypothesize based on our findings that such attraction does indeed exist, and we suggest that this attraction is probably the mechanism that explains the eventual assembly of a relatively normal IPL in lak mutants. Through AC-AC attraction, early errors could be corrected and later-arriving AC neurites could be correctly guided to the IPL (Fig. 8).
Together, our studies lead to a model of IPL formation in which ACs interact with GCs, and with each other, during assembly of the wild type IPL (Fig. 8). The interaction with GCs may be most important for positioning somata and orienting early outgrowth, whereas the attractive influence of other ACs and their neurites may stimulate AC innervation of the IPL.
ACs may drive IPL sublamination
We used line 220 to investigate how sublamination arises during
development. We found that the nascent IPL is not obviously stratified
(Fig. 2C) but over a period of
about 10 hours, distinct ON and OFF sublaminae form. Sublamination sweeps
across the retina in approximately the same spatiotemporal pattern that
characterizes the wave of AC neurogenesis, as has recently been suggested from
static images of the chick retina (Drenhaus
et al., 2003). This behavior suggests that sublamination may occur
at a fixed time after cell specification. Alternatively, there may be a
progressive mechanism for sublamination that propagates through the IPL in a
wave-like manner. In either case, our recordings have revealed that the IPL
undergoes significant morphogenesis between 60 and 70 hpf that results in the
formation of distinct sublayers.
We then asked if sublamination involves interactions between ACs and their
major postsynaptic target, the GCs. Our results demonstrate that GCs are
dispensable for sublamination of AC and BC projections, as previously
suggested by experiments in which GCs were removed postnatally in mammals
(Günhan-Agar et al.,
2000; Williams et al.,
2001
). Might other retinal cell types provide signals for AC/BC
sublaminar targeting? A mouse mutant lacking BCs shows normal AC sublamination
(Green et al., 2003
); it
therefore seems unlikely that ACs receive sublaminar cues from BCs.
Müller glia, which first differentiate around 60-70 hpf
(Peterson et al., 2001
), are
another potential source of sublamination cues. Our findings in lak
mutants suggest that AC neurites carry at least some targeting cues for their
synaptic partners: In areas of the mutant retina where sublaminar information
from ACs is degraded or lost, ON BCs fail to target correctly. Recent
molecular studies suggest that these AC-derived cues could take the form of
homophilic cell adhesion molecules, such as Ig-superfamily molecules or
cadherins (Yamagata et al.,
2002
; Masai et al.,
2003
).
Despite the fact that GCs are not essential for sublamination, the IPL is not completely normal in lak mutants - we found that sublamination is globally delayed, and that it fails altogether in local retinal patches. Such findings are consistent with the possibility that GCs provide one of several signals that influence sublamination. However, we think it is more likely that the presence of GCs is permissive: GCs may provide a scaffold that organizes the early IPL, setting the stage for a GC-independent sublamination process. According to this model, the initially-disorganized IPL that forms in the absence of GCs needs time to `recover' (self-correct by AC-AC interactions) before sublamination can start. That recovery does occur is shown by our time-lapse images - the unevenness and disorganization of the lak mutant IPL clearly improves with time. The retinal patches where sublamination fails may correspond to places where the early disorganization does not recover sufficiently to allow sublamination - for example, in places where an ectopic aggregate of AC processes has formed (see model in Fig. 8).
Targeting without a major target
Our finding that the presence of a major class of postsynaptic neurons is
dispensable for the proper arrangement of presynaptic terminals is not without
precedent. In the Drosophila optic lobe and the mouse
entorhinal-hippocampal projection, afferents depend on a transient
intermediate target rather than the eventual postsynaptic neuron for correct
sublaminar targeting (Huang and Kunes,
1996; Poeck et al.,
2001
; Suh et al.,
2002
; Del Rio et al.,
1997
). In contrast to these earlier studies, we show that ablation
of GCs disrupts the initial targeting of ACs, but that most of these errors
are later corrected. Our study therefore indicates that targeting cues are
provided both by postsynaptic partners and by presynaptic neighbors. The
picture that emerges is that of a delicately orchestrated interplay of
intrinsic programs and cell-cell interactions that act together (sometimes in
overlapping fashion) to bring axons into register in the target zone.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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