Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA
* Author for correspondence (e-mail: pmk{at}stowers-institute.org)
Accepted 8 October 2004
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SUMMARY |
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Key words: Chick, Neural crest, Cranial, Filopodia, Cell guidance, Confocal, Time-lapse imaging
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Introduction |
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Because neural crest cells arise, undergo extensive migration and
contribute to many different derivatives
(LeDouarin and Kalchiem,
1999), this system is an excellent model for studying induction,
cell guidance and cell differentiation
(Santagati and Rijli, 2003
).
Here, our focus is on the formation of the cranial neural crest cell migratory
pattern, which in vertebrates is a consistent, stereotypical pattern of three
segregated streams (Kulesa et al.,
2004
). From many cell labeling, tissue transplant and molecular
studies, several distinct hypotheses have emerged on how neural crest cell
streams form, but most agree that the pattern develops from a combination of
intrinsic and extrinsic factors (Trainor
and Krumlauf, 2000
; Halloran
and Berndt, 2003
; Graham et
al., 2004
). However, despite advances in identifying molecules
that induce premigratory cells and specify the fate of a cranial neural crest
cell (Knect and Bronner-Fraser,
2002
; Anderson,
2000
; Santagati and Rijli,
2003
), relatively little is known about the molecular mechanisms
that direct migrating cranial neural crest cells. Thus, one crucial step in
analyzing the pattern formation is to determine the extent to which cranial
neural crest cells interact with each other and the environment along the
migratory routes in vivo.
Cranial neural crest cells migrate along a dorsolateral route and, for the
most part, within one of three streams of cells that develop between the
neural tube and the branchial arches. The subregions of the neural tube from
which the majority of the neural crest cells emerge correlate with specific
segments [rhombomeres (r)] of the hindbrain, namely r1+r2, r4, and r6
(Lumsden and Keynes, 1989).
Early studies suggest that after receiving a particular molecular identity at
the neural tube, neural crest cells emerge and target a peripheral region with
a similar molecular identity (Hunt et al.,
1991
), carrying cues for the patterning of the arch from
neural-tube-derived signals (Noden,
1983
). The need for individual neural crest cell pathfinding in
this scenario is simplified if the neural tube controls cell exit points
(Lumsden et al., 1991
) such
that cells diffuse laterally from high populations to low populations in
segregated streams (LeDouarin,
1982
; Newgreen et al.,
1979
; Rovasio et al.,
1983
). Studies in chick
(Graham et al., 1993
;
Smith and Graham, 2001
) and
other amniotes (Knabe et al.,
2004
) show increased levels of cell death in specific rhombomeres,
particularly r3 and r5. However, there is no apoptosis specific to r3 and r5
in Xenopus, zebrafish, and mouse
(Schilling and Kimmel, 1994
;
Ellies et al., 1997
;
Hensey and Gautier, 1998
;
Del Pino and Medina, 1998
;
Kulesa et al., 2004
),
suggesting that cell death is not solely responsible for segregating the
streams. Thus, in a prepattern-type model the neural tube is thought to endow
destination instructions and control exit points such that there are few
directional cues necessary to produce the cranial neural crest cell
pattern.
In contrast to a prepattern hypothesis, studies of cell migratory behaviors
and the local environment adjacent to the neural tube suggest that the cranial
neural crest cell pattern emerges when cells encounter and respond to
environmental cues and interactions with other neural crest cells. Novel
culture and imaging techniques, combined with Nomarski optics and labeling of
premigratory neural crest cells, has allowed cell migratory behaviors to be
visualized in tissue culture (Abercrombie,
1970; Bard and Hay,
1975
; Erickson et al.,
1980
; Krull et al.,
1995
), in 2D and 3D gel substrates
(Newgreen et al., 1979
;
Rovasio et al., 1983
;
Thomas and Yamada, 1992
), in
whole embryo culture (Spieth and Keller,
1984
; Kulesa and Fraser,
1998
), and in ovo (Kulesa and
Fraser, 2000
). Analyses of cell movements suggest the mechanisms
that sculpt the pattern are more complex than would be expected from a purely
directed diffusion model and may include cell-cell and cell-environment
interactions in the form of chemotaxis, contact inhibition, contact guidance
and haptotaxis. From cell labeling studies in a variety of animal model
systems, it was learned that cranial neural crest cells emerge and emigrate
from all rhombomeres, rather than preferentially exiting from only the
even-numbered ones (Sechrist et al.,
1993
; Schilling and Kimmel,
1994
; Birgbauer et al.,
1995
; Trainor et al.,
2002
). Regions lateral to r3 and r5 inhibit neural crest cell
movements; cells stop and collapse filopodia or dramatically change direction
into a neighboring stream (Kulesa and
Fraser, 1998
). Avian grafting experiments suggest the
microenvironment adjacent to the neural tube may be important for maintaining
the proper segregation of the neural crest cell streams
(Farlie et al., 1999
). When
even-numbered quail rhombomeres are grafted lateral to and adjacent to chick
r3, cells from the transplant diverge toward the even-numbered streams rather
than migrate further laterally. There are clues that the repulsion is caused
by a secreted factor at the neural tube midline. When either the r3 chick
neuroepithelium or r5 surface ectoderm is removed, neural crest cells invade
the area immediately adjacent to r3 and r5, respectively
(Golding et al., 2002
;
Golding et al., 2004
). Thus,
in a self-organizing model, the neural crest cell pattern emerges from
multiple factors and regional differences.
The following study was guided by our interest in learning more about the nature of neural crest cell pathfinding. Here, we take advantage of the accessibility of chick embryos to perform direct observations. The proximity of the migratory routes (just underneath the surface ectoderm and relatively short time period over which neural crest cells migrate to the branchial arches) permit us to use a whole embryo explant culture method and perform hi-resolution static and confocal time-lapse imaging of individual cell migratory behaviors. We examined in detail to what extent neural crest cells interact with each other and the environment and whether this may influence cell directionality. Using a set of fusion protein constructs targeted to the cell membrane and nucleus, we were able to distinguish the lamellipodia and filopodia of individual neural crest cells. The time-lapse movies capture the dynamics of the cell-cell interactions and outline a chronology of the downstream movements of individual neural crest cells within the migratory streams exiting from r4 and r6. Some of the cell-cell interactions demonstrate obvious opportunities for cell-cell communication and directional guidance. Our data reveal an exciting level of detail to in vivo neural crest cell pathfinding and suggest that local and long-range cell-cell interactions play an important role in cell guidance.
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Materials and methods |
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Cell labeling
Embryos at 5-8 ss were injected with fusion protein constructs [GAP-43 EGFP
and H2B-MRFP (Okada et al.,
1999) (gifts from Dr Rusty Lansford/Caltech)] to label
premigratory cranial neural crest. After windowing the eggshell, a hole was
cut in the vitelline membrane above the neural tube at the cranial end using a
fine tungsten needle. In embryos labeled for fluorescence imaging, constructs
were injected into the lumen of the neural tube, filling the hindbrain region,
using a Picospritzer III (Parker Hannfin Corporation). A small amount (10
mg/ml) of Fast Green FCF (Fisher 42053) was added to the construct for easier
visualization during injection. The eggs were then electroporated using
platinum electrodes and an Electro Square Porator ECM 830 (BTX, a division of
Genetronics) with 20 V, 55 milliseconds pulse length, 5 pulses, at 1 second
intervals. Injected eggs were resealed with adhesive tape and re-incubated at
38°C. For static imaging, embryos were evaluated after 12-15 hours.
Embryos for time-lapse imaging were evaluated after 6 hours. A fluorescence
dissecting microscope (Leica MZFL III) was used to evaluate each embryo for
health, uniformity of labeling and brightness.
Static imaging
For static imaging, individual embryos were removed from the egg with paper
rings (Whatman #1), cleansed in Ringer's solution and placed dorsal side up
within a thin ring of high vacuum grease (Dow Corning 79810-99) on 22x75
mm microslides (VWR 48312-024). The embryo did not come in contact with the
vacuum grease. A small amount of Ringer's solution was pipetted away from the
embryo and the embryo positioning was adjusted with forceps. A 22x22 mm
glass coverslip (VWR 48312-024) was placed on top of the ring of grease,
creating a sealed, humidified chamber. The embryos were then imaged using one
of three microscopes (Zeiss Axiovert 200M, Zeiss LSM 510 META, and Zeiss LSM 5
PASCAL) and a wide range of objectives.
Time-lapse confocal imaging
Whole-embryo culture preparation
Whole-embryo explant cultures were prepared according to the method
described in Krull and Kulesa (Krull and
Kulesa, 1998). Briefly, embryos were removed from the egg by
placing an oval ring of filter paper (Whatman #1) around the circumference of
the embryo and then cutting around the outer edges of the ring. The ring with
the embryo attached was placed in Ringer's solution for rinsing. The paper
rings are approximately 1.5 cm along the major axis with holes wide enough to
provide ample space between the inner side of the rings and the embryos. This
method leaves the entire embryo, as well as the surrounding blastoderm,
intact. The excised embryos were cleansed of yolk platelets and India ink by
gently pipeting Ringer's solution across them with a P-200 pipetman.
Short-term, hi-resolution time-lapse imaging
For short-term (<5 hours), hi-resolution (>20x), in vivo
time-lapse imaging, embryos were mounted directly dorsal side down into glass
bottom microwell dishes (MatTek corporation, P35G-0-14-C). An embryo's
blastoderm was spread out using fine forceps. Excess Ringer's was removed to
stabilize the embryo while maintaining its 3D morphology. Once positioned, a
Teflon membrane ring was sealed on top of the embryo with a bead of silicone
grease. The membrane is designed to keep the embryo moist while allowing gas
exchange. The membrane ring was made from a Teflon membrane (YSI incorporated,
97L0038) stretched over a plastic ring (OD=1 inch, ID=7/8 inch) and sealed in
place using beeswax.
To maintain embryos at temperature, a single dish was placed on a heating plate (Lyon Electric, TX7115-020) kept at 38°C using a temperature controller (Cell Temp Bionomic Controller, BC-100). A heat sink compound (#276-1372A-RadioShack) was spread on the heating plate prior to the microwell dish placement to help heat convection to the plastic dish and its contents. Imaging was performed on an LSM 510 META (Carl Zeiss) or an Axiovert 200M inverted fluorescence compound microscope (Carl Zeiss) using a 40x (NA=0.75) plan-neofluar or 40x C-Apochromat W objective (Carl Zeiss). The GFP proteins were excited with the 488 nm laser line using the filter set (Chroma) intended for GFP. The RFP proteins were excited with the 543 nm laser line using the filter set (Chroma) intended for rhodamine. Images were recorded every 1-5 minutes and analyzed using either the AIM (Carl Zeiss) or Axiovision software (Carl Zeiss).
Long-term, low resolution time-lapse imaging
For long-term (>5 hours), low resolution (<20x), in vivo
time-lapse imaging, cultures were set up using Millicell culture inserts
(Millipore PICM 030-50) and six-well culture plates (Falcon 3046), similar to
the protocol described in Krull et al.
(Krull et al., 1995). The
culture insert membranes were precoated with 200 µl of fibronectin (Sigma
F-2006, diluted 1:50 in phosphate buffer) with the excess pipetted away. The
dorsal surface of the embryos was placed on the coated culture insert, leaving
the ventral surface exposed to the atmosphere. Excess Ringer's solution was
pipetted from the membrane surfaces at the rostral and caudal ends of the
explants, such that the flow of solution straightened the rostrocaudal axis of
the embryos. This naturally spread the explants without flattening the embryos
and mimicked the tension of the blastoderm normally created by the stretching
of the yolk sac. Each explant covered approximately two-thirds (
2.8
cm2) of the area of a culture insert. Each individual culture
insert was then placed in separate wells of a six-well plate. The membranes
were underlain with a Neurobasal medium (Gibco 21103-031), supplemented with
B27 (Gibco 17504-036) and 0.5 mmol/l L-glutamine (Sigma G-3126). Sterile water
was added to the any unfilled wells to minimize dehydration during time-lapse
acquisition. The edges of the six-well plate were then sealed with
parafilm.
Fluorescently-labeled explants were visualized using an inverted confocal microscope (LSM 510 META or LSM 5 Pascal; Carl Zeiss Inc.) using a 10x Neofluar (NA=0.30) lens with a zoom=2. This allowed observation of the entire r6 and r7 streams. For better image resolution, plastic was removed from the optical path by making holes in the bottom of the wells into which round 22 mm glass coverslips (VWR 48380-080) were sealed using silicone grease (Dow Corning 79810-99). The membrane of the Millipore culture insert becomes transparent when moist.
The microscope is surrounded by an incubating box fashioned from cardboard (4 mm thick) and covered with thermal insulation (Reflectix Co.; 5/16 inch thick). An enclosed heater (Lyon Electric Co. 115-20) maintained the cultures at 38°C for the duration of the time-lapse acquisition, with only mild temperature fluctuations. Images were recorded every 5 minutes and analyzed using the AIM software (Carl Zeiss).
Data analysis
Several features from the AIM software were used, including 2D and 3D
visualization, projection and depth coding (Carl Zeiss). The depth code
feature provided the ability to recognize which focal planes the cells were in
throughout a z-stack by assigning a color code to the pixel intensity
as a function of the z-depth. This was important for determining
whether two cells were in contact with one another. Stacks of images were
manipulated for analysis using VisArt (Carl Zeiss), Volocity (Improvision) and
Image J (NIH).
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Results |
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Neural crest cells within dense streams make numerous contacts with neighboring cells
Neural crest cells that traveled within the dense r4 and r6 streams
maintained numerous contacts with neighboring cells
(Fig. 1). The r4 stream was
composed of cells originating from r3-r5 and extended laterally to r4 around
the anterior portion of the otic vesicle
(Fig. 1A). The r4 stream was
densely packed with neural crest cells, as evidenced by the number of nuclear
stained cells (Fig. 1B). Many
of the cells within the r4 stream had a bipolar shape with two processes
extending in opposite directions, one directed toward the branchial arch
destination (Fig. 1B). Cells
closer to the front of the r4 stream had many more filopodia extended in a
variety of directions (described in a separate section below). Cells from the
r5 region are known to migrate and join the r4 and r6 streams. Near the mid-r5
region, cells migrated laterally and then turned in either the anterior or
posterior direction and moved in a perpendicular fashion to the r4 or r6
streams until making contact with cells in those streams
(Fig. 1C).
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An in-depth look at individual cells within migratory streams revealed the extent to which an individual cell had several contacts with neighboring cells (Fig. 1E, Fig. 2; see Movie 1 in the supplementary material). Color coding cellular features based on z-depth helped to confirm whether filopodial extensions between cells were in the same z-plane (i.e. in contact) with neighboring neural crest cells (Fig. 2). For example, two neighboring neural crest cells were in contact (Fig. 2A,B; arrow), but other filopodia may have actually been separated in z-height (Fig. 2A,C; arrowhead). This contact could not be determined precisely from a projected image (Fig. 1E). The ability to rotate and render a 3D z-stack of confocal images around different axes of rotation (Fig. 2D-F) revealed that there may be other filopodial processes emanating from cells that were not visible in 2D projections. For example, an individual neural crest cell may have been in contact with at least two neighboring fluorescently-labeled cells (Fig. 2D). The first contact is clearly visible (Fig. 2D-E, arrow). The second point of contact is via a branch of the filopodium (Fig. 2F, arrowhead) that is not visible in either the projected image (Fig. 1E) or the depth coding (Fig. 2C, arrowhead).
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Bipolar neural crest cells are found near the middle of migratory streams, hairy cells at the front
To determine whether there are differences in the proximal (back of the
stream) to distal (front of the stream) distribution of hairy versus bipolar
cells, we analyzed cell shapes in typical neural crest cell streams
(n=8). We then plotted the percent of hairy versus bipolar cells at
the front, middle and back of typical neural crest cell streams as a
percentage of the total number of cells in the stream
(Fig. 4A). We found that a
larger percentage of hairy cells tend to be at the stream front (distal)
(Fig. 4A). In a typical stream,
there were approximately three times as many hairy cells near the front
compared with bipolar cells. At the back portion of a stream, the percentage
of hairy and bipolar cells was nearly equal. By contrast, a much larger
percentage of bipolar cells was found in the midstream region; there were
three times as many bipolar cells as hairy cells in a typical migratory stream
(Fig. 4A). Thus, a typical
neural crest cell stream has the striking feature of hairy cells at the front
and bipolar cells distributed throughout the middle of the stream.
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Time-lapse analysis reveals that filopodia play a role in cell directionality
Contact with a lead cell often results in directional guidance by the follower cell
Long, extended filopodial processes at the leading edge of cells appeared
to act as active cell-cell contacts that influenced the direction in which a
cell subsequently moved. There were two typical neural crest cell migratory
behaviors that occurred when a follower cell contacted a downstream cell. In
one case, the filopodium made contact with the downstream cell, and then
retracted before the trailing cell began to move in the direction of the
downstream cell (Fig. 5). The
sequence of events was as follows. First, a cell extended numerous filopodia
in different directions throughout the local microenvironment
[Fig. 5 (t=0); red cell].
Second, an extended filopodium made contact with a neighboring cell
(Fig. 5; t=1 hour), and
elicited a response (Fig. 5;
t=1 hour 5 minutes). The trailing cell then retracted its filopodium
(Fig. 5; t=1.5 hours) and began
to move in the direction of the contact with the lead cell
(Fig. 5; t=3 hours).
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Long, thin cellular processes retain a contact between individual cells
As described previously, neural crest cells can be connected by thin, long
filopodia that are only 1-3 µm in diameter, but extend up to 100 µm in
length (Fig. 6; see Movie 5 in
the supplementary material). These long cellular processes typically did not
lie in a single focal plane, but transcended large x, y and
z distances, such that two cells within a migratory stream, although
located far apart, may have been connected
(Fig. 6B,C). Intriguingly, the
long filopodial connections wound in between neighboring neural crest cells
(Fig. 6B,C; arrow). An
individual cell may have maintained several concurrent connections between
neighboring cells, with connections of various lengths
(Fig. 6).
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Discussion |
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The observations of lamellipodial and filopodial dynamics between migrating
neural crest cells suggest a role for the cell-cell contacts in directional
guidance. Short, wide lamellipodial and short, thin filopodial contacts were
often exchanged between neighboring neural crest cells. A typical filopodial
extension from a trailing neural crest cell contacted, and tracked the back
end of a downstream cell (see Movie 4 in the supplementary material), or
retracted after contact and then re-extended, with the cell body migrating
toward the position of the contact (Fig.
5). These extensions appeared to provide a feedback to inform the
cell body what is in the local environment. The cell may then integrate the
information and alter its trajectory. We did not see any evidence of a
trailing cell nudging a lead cell forward, as judged by the lack of blebbing
at the front of the lead cell. Evidence of nudging has been reported in deep
cells of the fish Fundulus
(Tickle and Trinkaus, 1976).
Our data support the growing evidence for the role of filopodia in directional
guidance in other systems. During Drosophila neuromuscular synapse
formation, embryonic muscles at the target site extend dynamic actin-based
filopodia (myopodia) toward invading motoneurons
(Ritzenthaler and Chiba,
2003
). The myopodial contacts are thought to lure the motorneurons
to the proper synaptic targets. Also in Drosophila, during dorsal
closure the lead cells of an epithelial sheet send out numerous filopodia that
contact cells on the opposite side. When the assembly of protrusions is
inhibited, the adhesion and fusion of the opposing epithelial partners is
prevented, leading to segment misalignments
(Jacinto et al., 2000
). Thus,
our data indicate that local cell-cell contacts influence the migration of
neural crest cell direction. It will be interesting to test to what extent the
neural crest cells can pathfind when the protrusive activity of the filopodia
is modulated.
The long, thin filopodia that stretch between two neighboring neural crest
cells as the cells move apart suggests a role for filopodia in long-range cell
communication. We presented evidence that some neighboring cells maintain a
contact as one of the cells moves away (Figs
6,
7; see Movies 5, 6 in the
supplementary material). The process between the cells lengthens until it
breaks. In some cases, the trailing cell migrates toward the direction of the
fragmented contact, although we did not determine whether the cell precisely
follows the trail of the broken contact. Neural crest cells have been shown to
make numerous short (10-20 µm) filopodial contacts with neighboring neural
crest cells during migration in the chick cornea
(Bard and Hay, 1975) and in the
mouse gut (Young et al.,
2004
). In this case, the cell contacts are thought to play a role
in mediating the collective migration of the cells in chains. Our evidence of
cell-cell contact in neural crest cell streams includes much longer
connections (up to 100 µm) and is similar to the growing evidence in other
embryonic systems that non-neighboring cells form long-range contacts
(Rorth, 2003
;
Cohen, 2003
). The discovery and
analysis of long cellular extensions (cytonemes) in the Drosophila
imaginal disc revealed that cytonemes project from distant cells toward the
signaling centers of the disc
(Ramirez-Weber and Kornberg,
1999
). The cytonemes are thought to transport, deposit or retrieve
signaling molecules and play a role in pattern formation. This idea of
long-range cell communication was strengthened with recent work reporting that
during Drosophila sense organ development, long filopodia on a single
precursor cell convey signals at a distance to non-local neighbors to pattern
the field (De Joussineau et al.,
2003
). Previously, it had been thought that the signal to pattern
the sense organ was conveyed through the long-distance secretion of a
signaling protein. Thus, it will be interesting to investigate the
intracellular dynamics within the neural crest lamellipodia and filopodia
during cell-cell contact for the possible role in cell communication.
Our studies have shown that there is extensive detail in neural crest cell pathfinding in the form of short- and long-range cell-cell contacts in vivo, pointing to a diverse set of directional guidance mechanisms for neural crest cells (Fig. 8). The number and length of the contacts, in the form of lamellipodia and filopodia, vary depending on the cell's position within a migratory stream. Time-lapse confocal imaging reveals that the long-range cell-cell contacts, mediated by filopodial extensions, play a role in directional guidance by allowing trailing cells to follow downstream leaders. Short-range contacts between neighboring migrating neural crest cells appear to inform the cells of the position and number of neighboring cells. Long, thin filopodial connections allow two neighboring cells to remain in contact as the cells move apart. Our results support the hypothesis that a combination of intrinsic and extrinsic cues sculpts the cranial neural crest cell migration pattern, but that lamellipodia and filopodia play a critical role in neural crest cell pathfinding in the local microenvironment. Our evidence of neural crest cell short- and long-range cell communication parallels with data in Drosophila, mediated through myopodia and cytonemes, and opens several exciting lines of investigation. The neural crest cell-cell contacts may involve signaling to communicate positional information or allow cells of a similar fate to maintain a relationship. By contrast to our previous view of neural crest cell streams consisting of compactly shaped cells with lamellipodia and relatively short filopodia, our new perspective is that neural crest cell streams are very densely packed with lamellipodia and filopodia in constant cell contact, intertwined around local and non-local migrating cells. Further dissection of the function of the cell-cell and cell-microenvironment interactions will probably bring unexpected insights into how neural crest cells navigate.
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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/131/24/6141/DC1
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