Department of Biology, University of Washington, Seattle, WA 98195, USA
Author for correspondence (e-mail:
dww{at}u.washington.edu)
Accepted 2 June 2005
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SUMMARY |
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Key words: metamorphosis, pruning, local degeneration, EcR
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Introduction |
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Selective branch removal, or pruning, is found throughout the vertebrate
nervous system. Two of the best examples are the pruning of the sub-cortical
axonal projections of Layer 5 neurons of the cortex
(O'Leary and Koester, 1993)
and the pruning of motoneurons at neuromuscular junctions
(Keller-Peck et al., 2001
).
Both underscore the importance of this phenomenon in shaping neural circuitry,
but represent two extremes of pruning. Layer 5 cortical neurons remove large
identifiable lengths of axon from animal to animal; this has been termed
`stereotyped' pruning (Bagri et al.,
2003
). Pruning of motor axons in the vertebrate peripheral nervous
system, by contrast, is usually small scale (i.e. local) and stochastic in
nature.
The modes by which branches are eliminated also appear to vary. When motor
axon branches lose their contacts, they lift off the muscle, form a
`retraction bulb' and retreat backwards to the parent branch
(Keller-Peck et al., 2001). In
contrast, chick retinal axons that `overshoot' their targets within the optic
tectum appear to correct errors by local degeneration of the axon
(Nakamura and O'Leary, 1989
).
At present, we know little about the mechanisms that underlie either type of
pruning, and how different they truly are.
Axonal and dendritic pruning is especially pronounced in insects that
undergo complete metamorphosis. These insects build two distinct bodies: a
larval form for feeding and growing, and an adult form for reproduction and
dispersal. To transition between these two forms, the nervous system undergoes
dramatic changes which include the differentiation of adult-specific neurons,
the death of some larval neurons and remodeling of others
(Levine and Weeks, 1996;
Truman, 1996
). Studies of
central neurons in Drosophila have shown that pruning of axons and
dendrites can be blocked by interfering with either ecdysone signaling
(Schubiger et al., 1998
;
Schubiger et al., 2003
;
Lee et al., 2000
), TGFß
signaling (Zheng et al., 2003
)
or the ubiquitin proteasome system (Watts
et al., 2003
). Alongside the requirement for such intrinsic
factors, evidence is accumulating that extrinsic factors are also important
for pruning in some neurons. Glial processes infiltrate the neuropil just
prior to pruning of mushroom body
neurons, and afterwards contain
axonal debris (Watts et al.,
2004
). If glia are prevented from penetrating the neuropil, axon
pruning is blocked (Awasaki and Ito,
2004
).
The pruning of neuritic arbors is also seen in peripheral neurons in
Drosophila. During metamorphosis, the majority of larval sensory
neurons die but a small number persist to become functional adult neurons
(Williams and Shepherd, 1999).
Among those that survive are dendritic arborizing (da) sensory neurons
(Usui-Ishihara et al., 2000
;
Williams and Shepherd, 2002
).
The large, complex dendritic arbors of these neurons are completely removed
during early metamorphosis, before the cells elaborate their adult-specific
arbors (Smith and Shepherd,
1996
; Williams and Truman,
2004
).
Here we describe how da neuron dendrites are deconstructed during early metamorphosis and the contributions of intrinsic and extrinsic factors to this process. We find that microtubule cytoskeleton remodeling precedes the severing of proximal dendrites, and that this intrinsic mechanism can be blocked by the expression of a dominant negative ecdysone receptor. Our data also suggest that two other cell populations may participate in the deconstruction of the arbor. Phagocytic blood cells attack and sever intact branches, and the epidermis, the substrate over which these cells arborize, remodels whilst the neurons are pruning.
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Materials and methods |
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To disrupt ecdysone signaling, females of the genotype
UAS-EcR.B1-C655.W650ATP1-9
(Cherbas et al., 2003
) were
crossed with UAS-CD8::GFP; C161-GAL4,
UAS-CD8::GFP/TM6b. To block cell death, UAS-p35 females
were crossed to UAS-CD8::GFP; C161-GAL4,
UAS-CD8::GFP/TM6b. To image the epidermis, we used a genomic
rescue construct of histone H2A fused to GFP
(Clarkson and Saint, 1999
).
Staging of animals
Individual animals were collected at pupariation and maintained at
25°C. Staging was denoted as hours after puparium formation (h APF).
Immunocytochemistry and dye labeling
Dissection and immunohistochemistry were performed as described by Truman
et al. (Truman et al., 2004).
Primary antibodies included Rat anti-mCD8 (1:1000; Caltag Laboratories,
Burlingame, CA, USA), mAb 22C10 [1:500; Developmental Studies Hybridoma Bank
(DSHB), Iowa City, IA, USA] and anti-Cut (F2) (1:20, DSHB). Secondary
antibodies included; 488 Alexa Fluor anti-rabbit IgG (1:500; Molecular Probes,
Eugene, OR, USA) and Texas Red donkey anti-mouse IgG (1:500; Jackson
ImmunoResearch Laboratories, West Grove, PA, USA).
Acidic organelles were labeled with LysoTracker Red (Molecular Probes). A 1 nl bolus of 50% LysoTracker DND-99 was injected directly into prepupa 10 h APF using a pico-spritzer and a glass microneedle.
Image acquisition and processing
Confocal images were taken using a BioRad (Hercules, CA, USA) Radiance 2000
system equipped with a kryptonargon laser. Z-stacks were collected at
1.5 µm intervals (40x). For time-lapse imaging, individual prepupae
or pupae were mounted in an imaging chamber
(Williams and Truman, 2004)
and data acquired with the same Radiance system using a Ti:Sapphire laser
(Spectra-Physics, Fremont, CA, USA) set at 905 nm. Time-lapse frames consisted
of stacks of
25 sections at 1.5 µm intervals, acquired every 10
minutes. Stacks and movies were assembled in ImageJ, adjusted for brightness
and contrast using Photoshop (Adobe Systems, San Jose, CA, USA).
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Results |
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Deconstruction of the larval da sensory system
The five dorsal da neurons strongly labeled by C161-GAL4 show two
different fates: ddaA, ddaB and ddaF undergo cell death, whereas ddaD and ddaE
survive and remodel. Neither group shows any obvious change at 0 h APF (data
not shown). By 6 h APF the arbors of ddaF, ddaB and ddaA have largely
disappeared, although some fragments and blebs of GFP remain
(Fig. 1B). The corpses of the
cell bodies of these neurons can be seen in close proximity to the nerve.
The dendrites of ddaD and ddaE are still larval in their number and extent at 6 h APF. By 10 h APF (Fig. 1C), they have started to prune, with arbor loss more advanced in ddaE than ddaD. Filopodia are also found on the branches of both neurons. By 16 h APF, many of the higher order branches are lost, and detached branches appear to be separated from the main arbor (Fig. 1D). Filopodia are still found along the branches as the proximal regions begin to thin. By 24 h APF, the only remaining dorsal neurons that express the C161-GAL4 driver are ddaD, ddaE and dbd. Debris derived from pruned dendrites remain, and the cell bodies have filopodia-rich growth cones (Fig. 1E). Axons remain intact throughout and show no change in caliber.
The cellular dynamics of death
To understand the cellular mechanisms that bring about these dramatic
changes, we used in vivo time-lapse microscopy.
Fig. 2A shows frames from Movie
1 (see supplementary material) focusing on the distal arbors of 3 da neurons.
The neurons imaged are ddaF and ddaB, both of which die, and ddaD, which
survives and remodels.
At 0 h APF, the dendritic arbors are indistinguishable from their larval form. By 2 h APF, the dorsally projecting branch of ddaF develops constrictions and swellings which become more obvious `beads' by 3 h APF. Between 3 and 4 h APF, the branch severs at multiple sites, leaving blebs of GFP, which move away randomly. The other branches of ddaF follow the same sequence.
Between 3 and 4 h APF, the dendrites of ddaB also begin to generate constrictions and beads. Just after 5 h APF its branches sever, leaving only the arbor of ddaD intact by 6 h. The dying cells showed no filopodia. Beading appears to propagate through the arbor as a proximal to distal wave. Fig. 2B illustrates this wave in the dorsally projecting arbor of ddaF. At 1.5 h a distinct bead forms in the proximal region whereas the most distal part is intact. By 3 h the whole branch appears beaded, and by 4 h these beads move in random directions.
To visualize the microtubule cytoskeleton in these neurons, we used a tubulin GFP fusion protein, tub::GFP. At 0 h APF, the arbors have a uniform distribution of tub::GFP (data not shown). By 3 h APF, ddaF shows a redistribution of tub::GFP (Fig. 2C), with some regions losing tub::GFP while others accumulate it into beads. In contrast, the arbor of ddaD at 3 h APF still shows a uniform distribution of tub::GFP.
When p35 was expressed with C161-GAL4, cell death was blocked in ddaA, ddaF and ddaB (Fig. 2D). The distal dendrites of ddaF and ddaB did not bead and undergo degeneration by 5 h APF (Fig. 2D). At 18 h APF the cell bodies of these `rescued' da neurons were still evident, but their arbors had been largely removed (Fig. 2E) and abundant filopodia extend from all cells.
The cellular dynamics of pruning
To obtain a greater understanding of dendritic pruning in remodeling
neurons, we made time-lapse movies of ddaD and ddaE.
Fig. 3A shows selected frames
from a movie between 14 and 24 h APF (Movie 2 in supplementary material). At
14 h 10 min APF <50% of the arbors of ddaD and ddaE remain, with filopodia
found throughout. Severed branches, along with smaller fragments and blebs of
GFP are seen close by. By 17 h 10 min APF more branches are severed. The
primary branch of ddaE is also thinner than at 14 h 10 min APF. By 19 h APF
the major branches of both neurons have been severed from their cell bodies.
Between 19 and 24 h APF the separated branches decrease in length, thin and
form swellings.
|
Fig. 3C shows a ddaD branch undergoing a `distal' severing event. At 14 h 10 min the arbor appears stable, although there are swellings similar to those described above for proximal severing. Between 15 h 10 min and 15 h 20 min the branch severs, but shows no obvious thinning prior to this event.
Severed branches undergo fragmentation as illustrated in Fig. 3D. At 17 h 50 min the segment begins to thin and generate swellings. At 18 h 50 min the branch is beaded and is fragmenting into blebs. By 20 h most debris has disappeared.
In addition to severing and fragmentation, the branches of ddaD and ddaE can also undergo distal to proximal retraction. Fig. 3E shows a movie sequence in which retraction occurs in the proximal region of ddaD. At 16 h 30 min the primary branch of ddaD possesses a number of filopodia. Between 16 h 30 min and 20 h 00 min the branch retreats retrogradely towards the cell body. We saw retraction events in the distal regions of the arbor (branch labeled with asterisk in Fig. 3C retracts after severing event). While branches are retracting distally they may be severed at more proximal sites. Interestingly, severed branches can retract at both ends before undergoing fragmentation.
|
To examine the dynamics of the microtubule cytoskeleton, we made time-lapse movies of tub::GFP in ddaC (Movie 3 in supplementary material; Fig. 4D). At 0 h APF proximal arbors show uniform distribution of tub::GFP. By 2 h APF tubulin is redistributed and non-uniform. By 4 h APF, there is significant loss of tub::GFP in some regions while it is aggregated in large beads in other regions. These data show that a local loss of the microtubule cytoskeleton is associated with regions that undergo dendritic thinning.
|
When EcRDN is expressed in ddaA, ddaF and ddaB, cell death is blocked and the dendrites of these cells remain intact (Fig. 5B). Similarly ddaD and ddaE fail to prune properly; no severed branches or cellular debris are evident, and the proximal arbor does not `thin' (Fig. 5B). Instead the distal tips of the dendritic arbors of these neurons show bulb-like swellings, similar to retraction bulbs (Fig. 5E,F).
Time-lapse movies of EcRDN-expressing ddaD and ddaE (Movie 4 in supplementary material) reveal how branches are removed (Fig. 5G). At 16 h APF the arbor shows features described above with prominent retraction bulbs forming on the distal tips of dendrites. By 24 h 10 min APF the branches have shortened and the retraction bulbs have increased in size. The subsequent frames show a slow but steady progression of retraction towards the cell body. One branch in ddaE (Fig. 5G, 25 h 30 min) was seen to sever during retraction. This was the only severing event seen in five movies and it was not preceded by thinning. Thinning was never seen in the proximal region of the dendritic arbor, and even at 64 h APF EcRDN-expressing neurons still possess vestiges of larval branches and show no sign of adult outgrowth (data not shown).
Although the expression of EcRDN suppressed branch severing and local degeneration, the branches show a retrograde retraction. This retraction may be due either to an ecdysone-induced cellular program that is not blocked by EcRDN or to the neurons' response to changes in the epidermis, i.e. the substrate on which they arborize. Consequently, we examined the changes taking place in the epidermis during early metamorphosis.
|
The role of phagocytes in dendritic pruning
Besides their role in removing dead and dying cells during metamorphosis,
blood cells appear to be intimately involved in pruning dendrites. Migratory
cells labeled with GFP were found in most of the pupae with GFP-expressing da
neurons (`ph' in Fig. 3A; 24 h
00 min and Movie 2). Fig. 7A
shows a faintly labeled phagocyte entering the field from the top right. By 19
h 40 min a severed branch from ddaE shows beading and the phagocyte moving
into close proximity. At 19 h 50 min the branch fragments, and by 22 h 00 min
has disappeared and the GFP has been transferred to the phagocyte through
engulfment of labeled dendrite fragments. In
Fig. 7B such a phagocyte is
co-labeled with the acidotropic marker LysoTracker. These cells are not glia,
since they did not label with repo-GAL4 but instead express hemolectin-GAL4, a
phagocytic blood marker (data not shown).
Phagocytes also appear to play an active role in severing intact dendritic branches. Such attacks can occur at the distal tips of branches or at proximal sites near the cell body. Fig. 7C shows selected frames from a time-lapse sequence where the distal part of the primary branch of ddaE is being attacked by phagocytes. At 16 h 00 min a phagocyte enters the field from the top right and comes into contact with the distal tip of ddaE. By 17 h 00 min the distal part of the branch is gone and the phagocyte appears to be labeled with more CD8::GFP. At 17 h 00 min another phagocyte moves from the left field. This phagocyte attacks the end of the primary branch as it retracts.
Phagocytes can also attack proximal regions of the dendritic arbor. Fig. 7D shows a time-lapse sequence (movie 6) from 15 h 40 min APF focusing on interactions between a phagocyte and the proximal region of ddaE. Between 15 h 40 min and 17 h 30 min the phagocyte moves close to the pruning arbor. Between 18 h 10 min and 18 h 20 min the branch is severed where the phagocyte is located. A small bleb of GFP derived from the pruning branch appears to be internalized by the phagocyte at severing (18 h 30 min).
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Discussion |
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The dendritic arbors of neurons that die undergo local degeneration
Programmed cell death is a near ubiquitous phenomenon in the vertebrate
nervous system and essential for proper morphogenesis
(Burek and Oppenheim, 1996),
Surprisingly, most studies have focused on the cell body and paid little
attention to the fate of neuritic processes, even though the axons of most
projection neurons have reached their targets before the onset of cell death
(Perry et al., 1983
).
|
The destruction of peripheral processes has been most widely studied
following acute local trauma, where the axon branch distal to the lesion
undergoes a stereotyped degeneration by fragmentation, termed Wallerian
degeneration (Waller, 1850;
Beirowski et al., 2005
). There
are similarities between the degeneration of dendrites in our study and the
axon pathology seen in Wallerian degeneration. Firstly, in vitro models of
Wallerian degeneration reveal a proximal to distal spread of beading in the
separated distal neurites (Sievers et al.,
2003
). Secondly, the microtubule cytoskeleton is disrupted
following transection (Zhai et al.,
2003
) and the beaded morphology and redistribution of tubulin we
see in the da neuron dendrites resembles that seen in neurons treated with
microtubule depolymerizing drugs (He et al., 2003).
We found that the expression of EcRDN in neurons that were fated
to die blocked their death and prevented the local degeneration seen between 3
and 6 h APF (data not shown). This prompted us to ask whether the degeneration
normally observed is due to cell death directly or is the result of a
hormone-induced microtubule disassembly program running in parallel with cell
death. When we block cell death by expressing p35, the dendrites showed no
signs of degeneration by 5 h APF (Fig.
2D), yet by 18 h APF their arbor was largely removed
(Fig. 2E). Thus, with p35, the
dendrites of these doomed cells prune at the same time and in the same way as
ddaD and ddaE (data not shown), suggesting that under normal conditions, death
directly causes the dendrite degeneration observed in ddaA, ddaF and ddaB at
3-6 h APF. Similar observations made after killing da neurons by laser
ablation support this proposition
(Williams and Shepherd,
2002).
Pruning of da neurons by local degeneration and branch retraction
Local degeneration
Pruning of the dendrites of ddaD and ddaE starts between 6 and 10 h APF and
is largely complete by 24 h APF (Fig.
1). Our time-lapse movies reveal that dendrites undergo
deconstruction by two different cellular mechanisms: local degeneration
(Watts et al., 2003) and
branch retraction.
During local degeneration, branches are detached from the main arbor and
undergo fragmentation (Fig.
3A,E). Watts et al. (Watts et
al., 2003) found that axons and dendrites of mushroom body
neurons undergo local degeneration: between 4 and 6 h APF the processes
undergo blebbing, and by 8 h APF most of the dendritic arbor has been removed.
By 18 h APF the axons have fragmented, and during that time there was no
evidence to suggest that
neurons undergo retraction. Similarly, larval
projection neurons in the olfactory lobe undergo local degeneration
(Marin et al., 2005
).
A key step in local degeneration appears to be severing, which we were able
to visualize with time-lapse movies. Severing happens in one of two ways,
depending on position within the arbor. In proximal regions severing is
preceded by thinning (Fig. 3B).
Once a branch has thinned, it severs and the stump retracts, while the
separated arbor undergoes fragmentation. We found that beads adjacent to
thinning regions contain abundant Futsch and tub::GFP, whereas these are
lacking in the thinned regions, suggesting that the bulk of the microtubule
cytoskeleton is lost in these focal regions. This supports observations by
Watts et al. (Watts et al.,
2003) that myc-marked tubulin disappears from pruning
neurons before the axon is lost. Similarly, Bishop et al.
(Bishop et al., 2004
) showed
that just prior to shedding `axosomes', the proximal region of a retracting
axon becomes devoid of organized microtubules. When neurons express
EcRDN, the caliber of the proximal branches does not change,
suggesting that a redistribution of the microtubule cytoskeleton fails and
proximal severing is subsequently blocked.
The second type of severing occurs at more distal sites within the arbor (Fig. 3C). Here there is no thinning and only occasional beading; thus it is possible that different mechanisms are responsible for severing at distal sites. Nevertheless, distal severing is also suppressed in neurons that express EcRDN, suggesting that a destabilization is also important here.
After severing, both proximal and distally detached branches undergo
fragmentation. The branches thin whilst beads form along their length
(Fig. 3D) and break at multiple
sites, generating GFP blebs that are removed by phagocytes (see below).
Although most of the events observed in branch fragmentation during pruning
appear similar to those seen in the arbors of dying da neurons, an exception
is the appearance of filopodia. When we have simultaneously imaged the
microtubule cytoskeleton and the membrane in pruning neurons, we often find
filopodia are coincident with the areas of microtubule disorganization
(Fig. 4C). Bray et al.
(Bray et al., 1978) found
lateral filopodia when they applied colcemid to chick neurons in culture.
Filopodia do not appear on the arbors of the doomed cells as the cells are
undergoing programmed cell death. However, when cell death is blocked by
expression of p35 these cells later show abundant filopodia as their dendrites
are removed (Fig. 2E). This
suggests a fundamental difference between the dendrite removal observed during
the pruning of ddaD and ddaE, and that seen in cells that undergo programmed
cell death.
|
The dendrites of neurons expressing EcRDN form bulb-like
structures on their distal tips and produce few if any filopodia. Local
degeneration is blocked in these neurons, but they do eventually remove their
dendrites by retraction. A number of scenarios could explain this retraction
phenotype. It is possible that EcRDN does not block all ecdysone
signaling and so the cell intrinsic ecdysone pruning program has not been
entirely eliminated. It is also possible, but unlikely, that the
EcRDN results in a non-specific `neomorphic' as the specificity of
this reagent has been demonstrated by Cherbas et al.
(Cherbas et al., 2003). The
other possibility is that EcRDN completely blocks ecdysone
signaling and that another parallel intrinsic signaling pathway plays a role
in dendrite retraction. It is most likely that EcRDN completely
blocks ecdysone signaling and that the phenotype we see reveals extrinsic
factors that are important for dendrite pruning under wild-type conditions.
The neuron-specific expression of EcRDN means that ecdysone
signaling is only disrupted in the neuron and that the local environment can
undergo its normal hormone induced changes.
|
|
During early metamorphosis, glial processes infiltrate the mushroom body
neuropil when axons are undergoing local degeneration
(Awasaki and Ito, 2004;
Watts et al., 2004
).
Ultrastructure studies show that remnants of labeled neurons appear in these
glia (Watts et al., 2004
).
Awasaki and Ito (Awasaki and Ito,
2004
) also suggest that glia play an active role in the
destruction of the axons, since inhibiting endocytosis in the glia stopped
infiltration and blocked axon pruning. Our time-lapse data also reveal that in
the periphery phagocytic blood cells also attack intact dendritic branches. As
shown in Fig. 7B,C, phagocytes
both attack the distal tips of retracting branches and sever branches at
proximal positions. We do not observe the phenomenon of proximal severing by
phagocytes in every movie, which suggests that either severing can result from
other forces such as the shearing action of the reorganizing epidermis, or we
are not seeing all the phagocyte/neuron interactions because only a fraction
of phagocytes are labeled. Nevertheless our data shows that phagocytic cells
have an active role in the pruning of peripheral neurons.
During the clearance of apoptotic cells, phagocytes are known to recognize
specific `eat me' signals on the surface of a dying cell
(Savill and Fadok, 2000). Are
the phagocytes recognizing similar `bite me' signals on the arbors of the
pruning da neurons? Our observations suggest that they are targeting specific
regions of branches that show signs of destabilization. Targeting of
destabilized regions is especially obvious from the behavior of phagocytes
encountering neurons expressing EcRDN. These phagocytes ignore the
stable proximal regions of the arbor and instead only attack the distal tips
(Fig. 8).
Taken together, our data show that the pruning of the da neuron dendrites during metamorphosis is achieved by local degeneration and branch retraction. We propose that these phenomena are controlled by both intrinsic and extrinsic cellular mechanisms (Fig. 9). The initiation of pruning by the steroid hormone ecdysone results in the destabilization of the microtubule cytoskeleton within the dendritic arbor. This loss of microtubules results in the severing of the branches, either by attacking phagocytes or possibly from shearing forces produced in the remodeling epidermis. Severed dendritic branches undergo a distinctive `beading', similar to redistribution of the microtubule cytoskeleton found in the arbors of dying cells. Phagocytes are intimately involved in the fragmentation and consumption of severed branches, which leads to a rapid clearance of cellular debris.
We expect that the mechanisms described here are not restricted to the dendrites of Drosophila sensory neurons undergoing remodeling during metamorphosis. It will be very interesting to learn which mechanisms used by developing cells to remove their neuritic processes are employed by cells following trauma or the onset of disease.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/16/3631/DC1
* Present address: MRC Centre for Developmental Neurobiology, King's College
London, New Hunt's House, 4th Floor, Guy's Hospital Campus, London SE1 1UL,
UK
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