1 Division of Cell and Developmental Biology, University of Dundee, Wellcome
Trust Biocentre, Dow Street, Dundee DD1 5EH, UK
2 Department of Human Anatomy and Genetics, University of Oxford, South Parks
Rd, Oxford OX1 3QX, UK
* Author for correspondence (e-mail: k.g.storey{at}dundee.ac.uk)
Accepted 27 July 2005
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SUMMARY |
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Here, we show that many cells in the chick stem zone express both early neural and mesodermal genes; however, stem zone-specific gene expression can be induced by signals from underlying paraxial mesoderm without concomitant induction of an ambivalent neural/mesodermal cell state. The stem zone is a site of FGF/MAPK signalling and we show that although FGF alone does not mimic paraxial mesoderm signals, it is directly required in epiblast cells for stem zone specification and maintenance. We further demonstrate that caudal Hox gene expression in the stem zone also depends on FGF and that neither stem zone specification nor caudal Hox gene onset requires retinoid signalling. These findings thus support a two step model for spinal cord generation - FGF-dependent establishment of the stem zone in which progressively more caudal Hox genes are expressed, followed by the retinoid-dependent assignment of spinal cord identity.
Key words: Stem zone, Stem cells, Spinal cord, FGF, MAPK, Hox genes, Chick
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Introduction |
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In chick and mouse, the stem zone first becomes molecularly distinct just
prior to somitogenesis, when it expresses several transcription factors that
distinguish it from the rest of the neural plate. These genes include the
homeodomain-containing factor, Sax1
(Schubert et al., 1995;
Spann et al., 1994
) (see
Diez del Corral et al., 2002
),
and in the chick, the proneural gene homologue, cash4
(Henrique et al., 1997
).
Epiblast cells close to the primitive streak express Fgf8
(Crossley and Martin, 1995
)
and bra (Kispert et al.,
1995
; Kispert and Herrmann,
1994
) and once caudal regression of the primitive streak is under
way, expression of these genes spreads laterally into the morphologically
defined open neural plate in both chick and mouse
(Kispert and Herrmann, 1994
;
Kispert et al., 1995
;
Schmidt et al., 1997
). This
suggests that some cells in this region of the neuroepithelium co-express
early mesodermal and neural genes.
Signals from the regressing node can induce both cash4 and
Sax1 in the chick (Henrique et
al., 1997) however, ablation of the node does not result in loss
of Sax1 expression (Spann et al.,
1994
), suggesting that other tissues share this property. At later
stages, studies show that the paraxial mesoderm beneath the established stem
zone is indeed required for maintenance of cash4 and Sax1 in
the embryo (Diez del Corral et al.,
2002
). There is some evidence that FGF signalling accounts for
this maintenance signal from the mesoderm. The anterior primitive streak
expresses Fgf2, Fgf3, Fgf4, Fgf8, Fgf12, Fgf13 and Fgf18
(Boettger et al., 1999
;
Crossley and Martin, 1995
;
Karabagli et al., 2002
;
Mahmood et al., 1995
;
Ohuchi et al., 2000
;
Riese et al., 1995
;
Shamim and Mason, 1999
), and
most of these factors persist in the regressing streak and are present in the
stem zone itself (Fgf2, Fgf3, Fgf8, Fgf18) while Fgf8, Fgf10
and Fgf18 (Karabagli et al.,
2002
; Ohuchi et al.,
1997
) are also expressed by paraxial mesoderm. Furthermore, FGF4
or FGF8 can locally ectopically maintain expression of cash4 and
Sax1 as the spinal cord develops
(Bertrand et al., 2000
;
Diez del Corral et al., 2002
).
However, it is not known whether FGF acts directly on epiblast cells to
specify or maintain the stem zone.
FGF signalling has long been implicated in the generation of the vertebrate
body as disruption of this pathway results in failure to form this part of the
embryo (e.g. Amaya et al.,
1991; Draper et al.,
2003
; Griffin et al.,
1995
; Xu et al.,
1999
). The primary role of FGF signalling in mesoderm induction
has made it difficult to assess its direct requirement for induction of tissue
that depends on mesoderm derived signals (reviewed by
Bottcher and Niehrs, 2005
).
However, this pathway has been shown to initiate neural development in the
chick embryo, in mouse ES cells and most recently in the frog embryo
(Delaune et al., 2005
;
Streit et al., 2000
;
Wilson et al., 2000
;
Ying et al., 2003
) (reviewed
by Stern, 2005
). MAPK
activation downstream of FGF signalling is implicated in this step in the
chick (Eblaghie et al., 2003
)
and in the frog acts at least in part by interfering with BMP signal
transduction by inactivating the BMP intermediary protein Smad1
(Delaune et al., 2005
;
Pera et al., 2003
). MAPK
signalling is also required for mesoderm induction
(Saba-El-Leil et al., 2003
;
Umbhauer et al., 1995
;
Yao et al., 2003
) and recent
data suggest that low level FGF/MAPK may initiate neural development, while
higher levels promote mesoderm formation
(Delaune et al., 2005
).
Together, these studies indicate that serial FGF/MAPK mediated events may
underpin stem zone formation and raise the possibility that prolonged exposure
to such signalling is involved in specification and/or maintenance of this
cell population.
FGF signalling not only mediates cell fate specification in the early
embryo but also maintains an undifferentiated cell state in many cellular
contexts (reviewed by Diez del Corral and
Storey, 2004). During body axis extension, exposure to FGF
inhibits neuronal differentiation (Diez
del Corral et al., 2002
) and onset of ventral patterning genes
(Bertrand et al., 2000
;
Diez del Corral et al., 2003
;
Novitch et al., 2003
).
Furthermore, blocking FGF signalling also accelerates movement of cells out of
the stem zone into the transition zone, which eventually forms the neural tube
where neuronal differentiation commences
(Mathis et al., 2001
). These
findings indicate a role for FGF signals in keeping cells in an
undifferentiated, proliferative cell state and within the stem zone.
Importantly, the maintenance of this undifferentiated state may prolong the
period during which cells are able to respond to caudalising signals
(Mathis et al., 2001
;
Vasiliauskas and Stern, 2001
)
and may thereby account for the expression of progressively more caudal Hox
genes in the stem zone (Liu et al.,
2001
). These genes determine rostrocaudal character in the
emerging body axis (reviewed by Deschamps
et al., 1999
), so, for example, Hoxb8 expression
identifies the spinal cord and is expressed in the neural tube caudal to
somite 5 (Muhr et al., 1999
).
Interestingly, depending on context, many caudal Hox genes, including
Hoxb8 are induced by FGF or retinoic acid (RA) signalling
(Bel-Vialar et al., 2002
;
Liu et al., 2001
;
Muhr et al., 1999
;
Oosterveen et al., 2003
).
However, recent work shows that FGF and retinoid signalling are mutually
inhibitory in the extending body axis
(Diez del Corral et al.,
2003
), and raises the possibility that initiation of caudal Hox
gene expression in the stem zone under the influence of FGF switches to a
dependency on somite-derived retinoids in differentiating tissues (reviewed by
Diez del Corral and Storey,
2004
).
Here, we use a panel of neural, mesodermal and stem zone-specific marker genes to characterise the stem zone region. In vitro explant assays are used to identify tissues that specify this cell group and to assess whether stem zone specific gene expression can be induced independently of mesodermal gene expression. We further test whether FGF signalling is sufficient and/or directly required for specification and maintenance of this cell population. By assessing whether onset of stem zone and caudal Hox gene expression depends on the retinoid pathway, we also distinguish between the molecular mechanism underlying stem zone specification and maintenance, and that which assigns distinct rostrocaudal identities along the length of the spinal cord.
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Materials and methods |
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Whole embryo treatment with inhibitors
Chick embryos of appropriate stages were placed on 1.2 µm Millipore
filters (RTTP01300) in Opti.MEM (Gibco) supplemented with 5% calf serum
±SU5402 (Calbiochem) or PD184352 (a gift from P. Cohen, MRC PPU), and
cultured in separate wells (NUNC 4 plates) for 3 hours in CO2 (5%)
at 38°C.
FGF beads
Heparin-coated beads soaked in 50 µg/ml murine FGF8B (R&D Systems)
were grafted in New culture as described previously
(Storey et al., 1998).
In vitro explant culture
Explants of epiblast or paraxial mesoderm from stages HH3-8 were cultured
using standard techniques (Placzek and
Dale, 1999). Two explants were taken from each embryo and
comparisons were made between explant pairs (except where stated otherwise)
following exposure of one of the pair to any of the following: mouse FGF8B,
human FGF4 (R&D Systems) or SU5402 (concentrations in text); control
explants were cultured in DMSO only. Explants were processed as described
previously (Diez del Corral et al.,
2002
).
In vivo electroporation of dominant negative FGFR construct
A chick dnFGFR1 construct in which the truncated receptor (amino acids
1-425) is fused to eYFP in a Clontech vector (pEYFP-N1) (provided by
C. J. Weijer) (Yang et al.,
2002) or a control empty vector (pEYFP-N1) were introduced by
standard techniques for in ovo electroporation or using a custom made chamber
for transfection of embryos in EC culture (a gift from I. Mason, KCL, London)
using an IntraCell T160 or a BTX ECM 830 pulse generator.
Vitamin A-deficient quail embryos
Vitamin A-deficient quail embryos were provided by E. Gale and M. Maden
(KCL, London), and generated as described previously
(Dersch and Zile, 1993).
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Results |
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These gene expression patterns identify an enduring region of overlap between neural and mesodermal genes in the epiblast adjacent to the anterior primitive streak, which suggests that cells in this position have the potential to form mesodermal as well as neural tissue. The onset of Sax1 in epiblast flanking the streak at HH6-7 also raises the possibility that creation of an ambivalent neural/mesodermal cell state is a prerequisite for stem zone specification.
The spinal cord stem zone is specified by late primitive streak stages
To determine when the stem zone is specified, explants of HH3 epiblast [3c
explants (Muhr et al., 1999);
Fig. 2A] were cultured in
isolation and screened for expression of genes characteristic of the stem
zone. Sax1 and Hoxb8 are barely detected in 3c explants
after 24 hours and then in only a few cases (3/25; 2/10; respectively;
Fig. 2B,C). However, the early
mesodermal gene bra (8/13) and the pan neural markers Sox2
and Sox3 (11/15, 7/7 respectively) are detected in 3c explants after
18-24 hours (Fig. 2D,E; data
not shown). This indicates that 3c epiblast has early neural and mesodermal
cell characteristics, but has yet to receive signals that specify the stem
zone. We therefore next assessed explants of epiblast from the same position
(adjacent to the anterior primitive streak) at a later stage, HH4
(Fig. 2F). These 4c explants
express Sax1 (11/18) and Hoxb8 (13/16)
(Fig. 2G,H) within 24 hours,
indicating that signals that specify the stem zone have been received by the
late primitive streak stage.
Expression of stem zone specific genes is confined to neural plate also expressing early mesodermal genes
Importantly, like 3c epiblast, 4c explants express both the early
mesodermal marker gene bra (9/10 cases) and the neural marker
Sox2 (12/12) (Fig.
2I,J). A previous study has reported that explants of epiblast
adjacent to the primitive streak do not express bra
(Muhr et al., 1999). This
might be explained if explants in these two studies were taken from slightly
different regions. To address this possibility, we assessed marker gene
expression after 24 hours culture in HH4 epiblast explants taken either
adjacent to the primitive streak explant (4a) or lateral to this region (4b)
(Fig. 2K). Expression of all
marker genes is detected in 4a explants (Sax1, 7/13; Hoxb8,
8/11; bra, 8/8; Sox2, 6/7)
(Fig. 4L-O), but only the
pan-neural gene Sox2 is consistently expressed in 4b explants
(Sax1, 2/32; Hoxb8 0/10; bra, 3/30; Sox2,
23/26) (Fig. 4P-S). This may
explain the difference between our findings and those of Muhr et al.
(Muhr et al., 1999
), and
indicates that markers of the stem zone are detected only in epiblast that
expresses early mesodermal and neural genes, and/or that inclusion of
prospective paraxial mesoderm (as indicated by bra expression) is
required for Sax1 induction.
|
FGF signalling is not sufficient to specify the stem zone
We next tested whether FGF signalling is sufficient to mimic the signal(s)
provided by the paraxial mesoderm. FGF4 induces mesodermal genes in
extra-embryonic epiblast, but expression of stem zone genes takes place many
hours later, suggesting that FGF4 indirectly promotes formation of the stem
zone (Storey et al., 1998).
However, FGF4 induces Fgf8 in this assay
(Storey et al., 1998
) and FGF8
has been shown to be a rapid inducer of neural tissue
(Streit et al., 2000
) and so
one possibility is that FGF8 works directly to induce stem zone genes. Beads
soaked in FGF8 or control PBS washed beads were therefore grafted in contact
with HH3 extra-embryonic epiblast (Fig.
3A) and ectopic gene expression assessed at intervals. However, we
find that FGF8, like FGF4, first elicits expression of the mesodermal marker
bra (5/9) after 4 hours (Fig.
3B,C), and only somewhat later do we detect ectopic expression of
Sax1 (10/11) (10-12 hours) and cash4 (2/16) (16-18 hours)
(Fig. 3D; data not shown).
Using in vitro explant methods, we also assessed the ability of FGF4 to induce
Sax1 expression in 3c epiblast. These explants do not express
Sax1 after 24 hours of culture (22/25 as above) and addition of FGF4
does not elicit Sax1 expression (8/8) at 10 ng/ml (data not shown).
We also tested whether exposure to FGF4 or FGF8 promotes Sax1
expression in 4b explants. In nearly all cases, we found that neither FGF4 nor
FGF8 is able to elicit Sax1 in these 4b explants, although increases
in bra expression are found in control 4a explants and in a few 4b
explants in response to high concentrations of these factors, after 24 hours
(see Table 1; and
Fig. 3E-L). These findings
therefore demonstrate that FGF4 and FGF8 can promote mesodermal gene
expression in these assays but indicate that they are insufficient for
induction of stem zone marker genes. However, this does not exclude the
possibility that in addition to promotion of mesodermal character FGFs act on
the epiblast as necessary co-factors with other later mesoderm derived
signal(s) to specify the stem zone.
|
|
|
Cells mis-expressing dnFGFR1 tend to move out the stem zone, a phenomenon
that appears characteristic of cells lacking FGF signalling
(Mathis et al., 2001) and
which makes it difficult to target large groups of cells in this region. We
therefore also assessed expression of the Sax1 following a brief (3
hour) exposure to the FGFR antagonist SU5402 or the highly specific MEK
antagonist PD184352 (Davies et al.,
2000
) as this might provide a snapshot of FGF/MAPK requirement in
this tissue. In all cases, control DMSO and inhibitor treated embryos were
processed in parallel and reacted for the same period of time. Exposure of
embryos at HH9+-10 to SU5402 (60 µM) leads to loss of Sprouty2
expression (a known target of FGF signalling) and decreases Sax1
expression, while Sox2 expression is not reduced
(Fig. 5H-M; Table 2). Similarly,
Sprouty2 and Sax1 expression are depleted after exposure to
60 µM PD184352 (Fig. 5N,O;
Table 2), while again
Sox2 expression is not lost (Fig.
5P-S; Table 2). Together, these different experimental approaches indicate that FGF/MAPK
signalling is directly and specifically required in the epiblast to maintain
the stem zone.
|
|
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Discussion |
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The stem zone contains an ambivalent cell population
Analysis of the dynamics of marker gene expression in epiblast cells
adjacent to the primitive streak in the context of fate-mapping data provides
a detailed description of the stem zone and identifies an ambivalent cell
population within this region. We reveal an overlap between mesodermal and pan
neural gene expression at early streak stages in epiblast cells adjacent to
the primitive streak. In the mouse embryo single cell labelling has identified
epiblast cells at equivalent stages that contribute to both neural and
mesodermal lineages (Forlani et al.,
2003). By late streak stages in the chick, cell movement towards
the anterior primitive streak is much reduced
(Joubin and Stern, 1999
), but
this overlap between mesodermal and neural genes persists in epiblast close to
the anterior streak in the region where Sax1 expression commences.
Furthermore, cell labelling in the chick at HH4 and HH6-7+ reveals that some
cells remain in this region as it regresses caudally and that these cells can
contribute to mesodermal as well as neural lineages
(Brown and Storey, 2000
).
Clearly, although single cell labelling is required to determine whether these
resident cells are a mixture of mesodermal and neural precursors or a bipotent
cell population, the overlap of bra, Sox2 and Sax1 in this
region supports the existence of an ambivalent cell population. Furthermore,
HH6-7 stem zone explants cultured for a long period (48 hours) contain cells
expressing paraxis, a marker of differentiating paraxial mesoderm
(Diez del Corral et al.,
2002
), suggesting that bra expression in the stem zone is
indeed indicative of mesodermal potential and not just a marker of epiblast
cells close to the primitive streak.
At the ten-somite stage bra, Sox2 and Sax1 are expressed
in the stem zone (see Charrier et al.,
1999), while more rostrally transition zone cells express just
Sox2 and Sax1 (see Fig.
7). This distinction is consistent with cell labelling experiments
in the vicinity of the node at HH10, which show that here cells generate only
neural progeny (Mathis et al.,
2001
). Significantly, expression of Sip1, which directly
represses both bra and the epidermal gene E-cadherin
(Papin et al., 2002
;
Sheng et al., 2003
;
Van de Putte et al., 2003
), is
absent from the stem zone but is detected in the transition zone at HH10
(Sheng et al., 2003
) (K.G.S.,
unpublished), an observation that further supports the multipotency of cells
in the stem zone. The evidence for neural stem cells in the mouse stem zone
comes from an experiment in which only cells expressing a reporter gene under
control of a neural specific promoter are labelled
(Mathis and Nicolas, 2000
) and
so clonal contribution to both neural and non-neural tissue has yet to be
determined. It is therefore currently not known whether generation of the
spinal cord depends on stem cells that give rise to mesodermal and neural
progenitors located in the stem zone or if this relies on a distinct neural
stem cell population. An interesting possibility is that ambivalent stem zone
cells may be part of the self-renewing cell group identified in the mouse tail
bud at the junction between the node and anterior primitive streak, which
gives rise to both neural and paraxial mesodermal derivatives, although these
cells also retain the ability to form notochord
(Cambray and Wilson,
2002
).
Specification of the stem zone
Identification of an ambivalent cell population in the stem zone led us to
examine the specification and regulation of this region, and to assess whether
this specification step involves the creation of an ambivalent cell state. We
found that the stem zone, as indicated by expression of Sax1 and
Hoxb8, is specified by late primitive streak stages via signals
presented by caudal paraxial tissue. This differs from a previous report that
Hoxb8 is induced by rostral paraxial tissue taken from above the
level of the primitive streak (Muhr et
al., 1999), but this probably reflects differences in
Hoxb8 regulation in distinct domains (see below). Using lateral
neural plate (4b) explants, which express Sox2 but neither
Sax1 nor bra, we found that caudal paraxial mesoderm could
induce stem zone character without concomitant expression of bra.
This strongly suggests that it is possible to establish stem zone character
without first passing through an ambivalent mesodermal/neural cell state.
However, expression of Sox2 and Sax1 is indicative of
transition zone cells and as noted above, we have yet to ascertain whether a
purely neural cell population is sufficient to generate the entire spinal cord
or whether bipotent cells in the stem zone underlie this activity.
|
As FGF signalling is not sufficient for stem zone specification other
signals provided by the caudal paraxial mesoderm must be involved. These
include WNT proteins (Nordstrom et al.,
2002) and TGFß family members
(Liu et al., 2001
). Here, we
have focussed on the retinoid pathway, as the retinoid synthesising enzyme
Raldh2 is initially expressed in the caudal paraxial mesoderm
(Swindell et al., 1999
) just
prior to Sax1 onset. However, we find that onset of both
Sax1 and Hoxb8 appear at the normal time in vitamin
A-deficient embryos, indicating that retinoic acid is not necessary for stem
zone specification. By contrast, during normal development, the Sax1
expression domain appears to expand rostrally, coincident with restriction of
Raldh2 expression to rostral paraxial mesoderm. This rostral retreat
of Raldh2 reflects the persisting expression of Fgf8 and
bra in the stem zone and in paraxial mesoderm cells emerging from the
primitive streak and is driven by the ability of FGF signalling to inhibit
onset of Raldh2 (Diez del Corral
et al., 2003
).
|
FGF signalling declines as cells move from the stem zone to transition zone
and is driven by the ability of retinoic acid to attenuate Fgf8
levels (Diez del Corral et al.,
2003). In the frog, high FGF signalling induces mesoderm, while
lower levels promote neural tissue
(Delaune et al., 2005
) and a
similar conclusion can be inferred from data in the chick
(Eblaghie et al., 2003
;
Storey et al., 1998
;
Streit et al., 2000
) (this
paper). This finding fits nicely with the loss of bra expression as
cells leave the stem zone and encounter retinoic acid. Furthermore, ectopic
caudal neural tissue forms at the expense of mesoderm in Cyp26a
mutant mice and following application of exogenous retinoic acid
(Abu-Abed et al., 2001
;
Sakai et al., 2001
) (reviewed
by Maden, 2002
) and this
phenotype is also observed in embryos lacking FGF signalling
(Ciruna et al., 1997
).
Consistent with this, retinoid deficient embryos have a strikingly narrow
neural tube, suggesting that fewer cells are assigned to a neural fate when
retinoid levels are low (Diez del Corral
et al., 2003
; Maden et al.,
1996
; Molotkova et al.,
2005
; Wilson et al.,
2003
). Retinoic acid, by controlling Fgf8 levels, may
thus also help to resolve mesodermal versus neural cell fates in the extending
body axis.
Stem zone specification and maintenance is distinct from assignment of spinal cord character
Previous work has analysed the regulation of Hoxb8 as a marker of
spinal cord identity (Muhr et al.,
1999). This involved examination of the signalling pathways
required for regulation of Hoxb8 by rostral, but not caudal, paraxial
mesoderm. This rostral mesoderm does not express FGFs, but synthesises
retinoic acid (Berggren et al.,
1999
; Swindell et al.,
1999
), and Muhr and colleagues demonstrated a requirement for the
retinoid pathway, but not FGF signalling, for induction of Hoxb8 by
rostral paraxial mesoderm. Hoxb8 is first expressed in the stem zone
at HH8+ (Fig. 1) and, unlike
Sax1, persists in the neural tube, defining neural tissue caudal to
somite 5. It also later extends rostrally into the posterior hindbrain where
it again relies on retinoic acid
(Oosterveen et al., 2003
).
However, exposure of the early embryo or explanted caudal neural tissue to FGF
promotes Hoxb8 expression
(Bel-Vialar et al., 2002
;
Dasen et al., 2003
;
Liu et al., 2001
), suggesting
that an initial phase of Hoxb8 expression is responsive to FGF
signalling. We have shown here that Hoxb8 induction by caudal
paraxial mesoderm is dependent on FGF signalling and that brief exposure of
the whole embryo to an FGFR inhibitor attenuates Hoxb8 expression in
caudal regions. Furthermore, as noted above, onset of Hoxb8 in the
stem zone does not require retinoid signalling. These findings demonstrate
that distinct molecular mechanisms underlie the specification and maintenance
of the stem zone and assignment of a fixed spinal cord identity and support a
two step model for spinal cord generation, based on the opposition of FGF and
retinoid signalling pathways in the extending body axis
(Diez del Corral and Storey,
2004
). This involves the production of new tissue in the stem zone
under the influence of FGF signalling, where progressively more caudal Hox
genes are expressed. This is followed by somite-derived retinoid signals,
which attenuate FGF signalling as cells leave the stem zone and enter the
forming neural tube. Retinoid signalling then drives differentiation,
including assignment of rostrocaudal identity, as the progressive onset of
more caudal Hox genes ceases when cells form the neural tube.
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ACKNOWLEDGMENTS |
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