COUP-TFII is essential for radial and anteroposterior patterning of the stomach
Norio Takamoto1,
Li-Ru You1,
Kelvin Moses1,
Chin Chiang2,
Warren E. Zimmer3,
Robert J. Schwartz1,4,
Francesco J. DeMayo1,4,
Ming-Jer Tsai1,4,* and
Sophia Y. Tsai1,4,*
1 Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030, USA
2 Department of Cell Biology and Neurosciences, University of South Alabama,
Mobile, AL 36688, USA
3 Department of Cell and Developmental Biology, Vanderbilt University Medical
Center, Nashville, TN 37232,USA
4 Developmental Biological Program, Baylor College of Medicine, Houston, TX
77030, USA
*
Authors for correspondence (e-mail:
stsai{at}bcm.tmc.edu;
mtsai{at}bcm.tmc.edu)
Accepted 1 March 2005
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SUMMARY
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COUP-TFII, an orphan member of the steroid receptor superfamily,
has been implicated in mesenchymal-epithelial interaction during
organogenesis. The generation of a lacZ knock-in allele in the
COUP-TFII locus in mice allows us to use X-gal staining to follow the
expression of COUP-TFII in the developing stomach. We found
COUP-TFII is expressed in the mesenchyme and the epithelium of the
developing stomach. Conditional ablation of floxed COUP-TFII by
Nkx3-2Cre recombinase in the gastric mesenchyme results in
dysmorphogenesis of the developing stomach manifested by major patterning
defects in posteriorization and radial patterning. The epithelial outgrowth,
the expansion of the circular smooth muscle layer and enteric neurons as well
as the posteriorization of the stomach resemble phenotypes exhibited by
inhibition of hedgehog signaling pathways. Using organ cultures and
cyclopamine treatment, we showed downregulation of COUP-TFII level in the
stomach, suggesting COUP-TFII as a target of hedgehog signaling in
the stomach. Our results are consistent with a functional link between
hedgehog proteins and COUP-TFII, factors that are vital for
epithelial-mesenchymal interactions.
Key words: Nuclear orphan receptor, Sonic hedgehog, Organogenesis, Stomach, Mouse, Nr2f1, Nr2f2
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Introduction
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Gut development depends upon crosstalk between endoderm and splanchnic
mesenchyme cell layers along the anteroposterior axis, resulting in the
subdivision of the gut into foregut, midgut and hindgut
(Kedinger et al., 1998b
;
Kedinger et al., 1998a
;
De Santa Barbara et al., 2002
).
The stomach becomes specified at the caudal end of foregut and emerges as a
bulge of the developing GI tract (Kaufman
and Bard, 1999
). The stomach then undergoes progressive remodeling
and differentiation in a regionally specific fashion. The detailed genetic and
molecular pathways that direct gastric organogenesis are still unknown, but it
is clear that patterning of the GI tract requires elaborate cellular
communication between the epithelium and mesenchyme cell layers
(Fukuda and Yasugi, 2002
).
Sonic hedgehog (Shh) patterns a variety of embryonic tissues,
including the fore-stomach epithelium by signaling to the mesenchyme prior to
organ regionalization (Chiang et al.,
1996
). Shh-null mice exhibit inappropriate expression of
alkaline-phosphatase (EAP) in the epithelium of the hind-stomach
(Ramalho-Santos et al., 2000
),
which is consistent with the stomach epithelium acquiring an intestinal or
`posteriorized' character. Conversely, the stomach of the activin receptor IIA
and B compound mutant (ActRIIA/B mutant;
Acvr2a/Acvr2bMouse Genome Informatics) exhibits posterior
extension of the fore-stomach epithelium with expansion of Shh
expression (Kim et al., 2000
),
a process referred to as `anteriorization'. These data suggest that
Shh expression in the fore-stomach acts to induce and/or maintain
non-intestinal character, resulting in the development of gastric epithelium
(Ramalho-Santos et al.,
2000
).
COUP-TF proteins are nuclear orphan receptors, highly conserved across
species. Two members have been identified in mice, COUP-TFI
(Nr2f1Mouse Genome Informatics) and COUP-TFII
(Nr2f2Mouse Genome Informatics). The temporal and spatial
expression pattern of COUP-TFII in mesenchyme led us to hypothesize
that COUP-TFII plays a role in mesenchymal-epithelial interactions
during organogenesis (Tsai and Tsai,
1997
). In the developing neural tube, Shh has been shown
to regulate COUP-TFII expression during the differentiation of
motoneurons (Lutz et al.,
1994
). We have also identified a 5'-regulatory element that
mediates Shh stimulation of COUP-TFII expression
(Krishnan et al., 1997a
;
Krishnan et al., 1997b
).
COUP-TFII is likely to be a downstream target of Shh
signaling, and the requirement for Shh in gastric organogenesis led
us to infer that COUP-TFII may play a role in stomach
development.
To circumvent the early embryonic lethality of the COUP-TFII-null
mutation and to investigate its function during gastric organogenesis, we
generated a conditional null mutant of COUP-TFII using the
Cre/LoxP system. Nkx3-2 (Bapx1) is a
homeobox-containing gene (Tribioli et al.,
1997
) that is coexpressed with COUP-TFII in the stomach
primordium; thus, Nkx3-2Cre knock-in recombinase was used
to ablate COUP-TFII in the gastric mesenchyme. The stomachs of
conditional mutant mice exhibited dysmorphogenesis accompanied by
abnormalities of both compartmentalization and radial patterning,
demonstrating a functional link between Shh and
COUP-TFII.
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Materials and methods
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Generation of conditional mutant of COUP-TFII
Genomic DNA fragments, 7.2 kb BamHI-SalI, 1.4 kb
SalI-XbaI and 1.4 kb XbaI-EcoRI from Q#10
clone and a 2.6 kb EcoRI-BamHI fragment from D-3.6RI clone
containing COUP-TFII locus as described
(Qiu et al., 1995
) were
subcloned into pBlueScriptII KS(+). The first loxP site was inserted into
BglI site of 7.2 kb BamHI-SalI fragment and the
NsiI site on the 2.6 kb EcoRI-BamHI fragment was
modified with XhoI linker. These four genomic fragments were
reconstructed, and XhoI site on the 2.6 kb
EcoRI-BamHI region was modified with linker containing
ClaI site at the 3' end. pNeoTKLoxP, a positive-negative
selection cassette, was modified and ligated with lacZ from pDP46.21.
This NeoTKLoxP-lacZ construct was inserted into
XhoI-ClaI linker at 3'-UTR. 5'-SalI
site of NeoTKLoxP-lacZ was destroyed by ligation with XhoI,
and ClaI site and NotI site were blunt-end-ligated after
fill-in reaction. Targeting construct consists of 4 kb 5'-arm, 6.5 kb
floxed COUP-TFII locus, 8.5 kb NeoTKLoxP-lacZ and 2 kb 3'-arm,
a total size of
24 kb was linearized with NotI at the 3'
end. Homologous recombination was performed in AB1.2 cells
(Qiu et al., 1997
) and ES cell
clones were screened by two rounds of Southern blot analysis. Correctly
targeted clones were first identified by screening XbaI digest with
5'-external and 3'-external probes (5'-ext and 3'-ext,
respectively) and subsequently transfected with a Cre recombinase expression
vector followed by screening HindIII digests with a 5'-internal
probe (5'-int) and XbaI digest with a 3'-external probe.
Chimeric animals were generated by microinjection and germline transmission
was achieved by crossing with C57Bl/6 wild-type female. Thereafter, mutant
mice carrying floxed allele were maintained in 129/B6 mixed background.
Specific primers were designed for floxed COUP-TFII genotyping by
PCR. The primer sequences were NT1
5'-CAGTCGCCTCTCCTTCTCCTTCTCC-3', NT2
5'-CATCCGGGATATGTTACTGTCCGG-3', NT3A
5'-TTCTGGTCTTCACCCACCGGTACC-3' and NT6A
5'-TGGGGAAGCTAAGTGTTGTAGTGATTCC-3'. The sizes of the PCR product
are 785 bp for wild type and 394 bp for floxed allele.
Nkx3-2Cre knock-in animal was generated by homologous
recombination in ES cells (K.M., W.E.Z. and R.J.S., unpublished). Cre
recombinase was inserted in-frame into the exon1 of Nkx3-2 locus. No
overt phenotype was found in heterozygous animals.
X-gal staining
X-gal staining was performed according to the published methods
(Hogan et al., 1994
). For
whole-mount staining, embryos (up to E10.5) were dissected and fixed in 4%
paraformaldehyde, then stained in X-gal staining solution at room temperature.
Histological sections of whole-mount stained embryos were processed in
Histoclear, embedded in paraffin, and sectioned at 10-12 µm. Sections were
cleared with Histoclear, rehydrated and counterstained with Eosin, and mounted
with Permount. Larger embryos (
E12.5 or later) and postnatal samples were
cryoprotected with 20% sucrose/PBS solution after fixation with 2%
paraformaldehyde and embedded in OCT compound. Cryostat sections (16 µm)
were briefly fixed in 2% paraformaldehyde, stained in X-gal staining solution
and counterstained with Eosin.
Histological analysis
Tissues were fixed in 4% paraformaldehyde, embedded in paraffin and
sectioned at 5 or 7 µm. Hematoxylin and Eosin staining was carried out
according to the regular protocol. For immunohistochemistry, paraffin sections
were dewaxed, rehydrated and incubated with primary antibodies. Antibodies
against human CK14 (1:500) was a gift from Dr Roop (Baylor College of
Medicine, Houston, TX). Polyclonal anti-serum against PGP9.5 was from Chemicon
(1:1000). Antibodies against TUJ1 (Convance, 1:1000), GATA4 (Santa Crutz,
1:400), H+/K+-ATPase ß-subunit (Affinity
BioReagents, 1:500) were used in immunostaining. Texas Red-tagged
Griffonia simplicifolia II (GSII) lectin was used to stain the
stomach tissue. Positive staining for CK14 and PGP9.5 was visualized by using
biotinylated secondary antibody and streptavidin-horseraddish peroxidase
conjugate, and NovaRed (Vector) as a chromogen. SMA-
, TUJ1, GATA4 and
H+/K+-ATPase ß-subunit were visualized by using
biotinylated secondary antibody and streptavidin-horseraddish peroxidase
conjugate, and DAB (Vector) as a chromogen. Monoclonal anti-
-smooth
muscle actin (SMA-
, 1A4, 1:1000) was purchased from Sigma-Aldrich and
immunostaining was performed using Mouse-On-Mouse kit (Vector) according to
manufacturers instruction, and detected with AlxaFluor488 (Molecular Probes).
For ultrastructural study of the disorganized smooth muscle layer, 0.5 µm
semi-thin sections were stained with Toluidine Blue. Endogenous
alkaline-phosphatase staining was performed as previously described
(Ramalho-Santos et al., 2000
;
Aubin et al., 2002
) and
counterstained with Methyl Green. The glycoconjugated production in parietal
cells of the stomach was marked by DBA lectin. Whole-mount in situ
hybridization for COUP-TFII was carried out as described
(Qiu et al., 1997
;
Pereira et al., 1999
). Probe
for Shh was a gift from Dr M. P. Scott (Stanford University, CA). For
section in situ hybridization of Shh, E11.5 embryos were
cross-sectioned and Shh expressions in the fore-stomach were detected
using 35S-UTP labeled probes. Positive signals were pseudo-colored
(red) and overlaid on bright field image of Hematoxylin staining as described
(Pereira et al., 1999
).
Explants culture of embryonic foregut
Foreguts were dissected from E10.5 lacZ knock-in heterozygous
embryos and cultured on tissue-culture insert (Corning) with defined
serum-free medium (Opti-MEMI, Invitrogen Life Technologies). Explants were
incubated with vehicle (DMSO), 1, 5 and 10 µM of cyclopamine (Toronto
Research Chemicals) for 48 hours, and fixed with 2% paraformaldehyde for
whole-mount lacZ staining. Explants from wild-type embryos were fixed
with 4% paraformaldehyde and used for whole-mount in situ hybridization.
 |
Results
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Expression of COUP-TFII in the stomach
To facilitate the profiling of COUP-TFII gene expression during
development, we generated a targeted vector according to a scheme diagramed in
Fig. 1A. This targeting vector
was introduced into ES cells together with Cre recombinase. Upon recombination
between the first and the last LoxP sites, COUP-TFII was
deleted and the expression of lacZ reporter, inserted into the
5' untranslated region of COUP-TFII locus, was activated. The
ES cells were subsequently injected into blastocysts to generate lacZ
knock-in alleles in mice. lacZ gene expression is under the control
of COUP-TFII promoter and its expression recapitulates the expression
of the endogenous COUP-TFII gene. Using this lacZ knock-in
allele, we examined the expression pattern of COUP-TFII during gut
development.

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Fig. 1. Expression of COUP-TFII in the stomach using lacZ
knock-in model. (A). Generation of the lacZ knock-in allele and
generation of floxed COUP-TFII allele. Using homologous
recombination, a targeting construct containing nuclear lacZ, Neo/TK
and LoxP sites were inserted into the genomic COUP-TFII
locus, generating a targeted allele in ES cells. Treatment with Cre
recombinase and FIAU selection resulted in a lacZ knock-in allele in
which lacZ gene expression was controlled by the endogenous
COUP-TFII promoter when recombination took place between the first
and the third loxP sites of the targeted allele. In addition, floxed
COUP-TFII ES clones that retains COUP-TFII locus but lacks
selection markers were generated when recombination took place between the
second and the third loxP sites. B, BamHI; H, HindIII; S,
SalI; X, XbaI. (B) Cryostat sections of E12.5 heterozygous
COUP-TFII/lacZ knock-in embryo were stained (for 2 hours) for
lacZ activity. There is relatively high expression in the mesenchymal
cells just adjacent to the epithelium. (C). The stomach from a 3-day-old
heterozygous knock-in animal was dissected and whole-mount X-gal staining was
performed. The boundary between stomach and duodenum is indicated by
arrowhead. (D) A cryostat section of stomach from adult heterozygous knock-in
animal was stained for lacZ activity (blue) and counterstained with
propidium iodide (red). DBA lectin immunostaining denotes the parietal cells
(green). There is strong X-gal staining in the base layer and negligible
staining in the surface pit layer of the adult Zymogenic unit. m, mesenchyme;
e, epithelium; fs, fore-stomach; hs, hind-stomach; d, duodenum; oe,
esophagus.
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At embryonic day (E) 12.5, X-gal staining was detected in both the
epithelium and mesenchyme of the stomach. Brief staining of cryostat sections
revealed that mesenchymal cells adjacent to the endoderm had higher expression
of COUP-TFII than did distal mesenchymal cells, suggesting that a
potential diffusible molecule, secreted from the epithelium might regulate
COUP-TFII expression in the mesenchyme
(Fig. 1B). At postnatal day 3,
COUP-TFII/lacZ was detected throughout the stomach
(Fig. 1C), and was sharply
downregulated in duodenum (red arrowhead indicates junction between stomach
and duodenum). COUP-TFII/lacZ was also expressed in the glandular
epithelium of adult stomach. Adult glandular epithelium forms tubular
structures, each of which is referred to as a zymogenic unit, which can be
subdivided into four regions; a surface pit, an isthmus, a neck and a base
that is located proximal to the sub mucosa
(Karam and Leblond, 1993
).
Strong lacZ staining was evident at the base, and much lower at surface pit
layer (Fig. 1D). To ensure
COUP-TFII is also expressed in the parietal cells, we used DBA lectin for
immunostaining. As shown in Fig.
1D, X-gal and lectin-positive green cells colocalized, indicating
that COUP-TFII is expressed in the parietal cells of the Zymogenic unit. The
expression pattern of COUP-TFII in the Zymogenic unit mimics the reported
expression pattern of Shh in mouse stomach
(van den Brink et al., 2001
).
Thus, COUP-TFII is abundantly expressed from an early developmental
stage in both epithelium and mesenchyme of the stomach in parallel with
Shh.
Nkx3-2Cre recombinase ablates COUP-TFII in the gastric mesenchyme
To examine the role of COUP-TFII in gastric development, we chose
to ablate the COUP-TFII gene in a tissue-specific manner using the
Cre/LoxP system. The targeting vector depicted in
Fig. 1A, was introduced into ES
cells. After excision of the Neo-TK cassette by Cre-based recombination, ES
cells harboring the conditional allele were generated as depicted in
Fig. 1A and used to generate
floxed COUP-TFII mice. An Nkx3-2Cre mouse line
was also generated (K.M., W.E.Z. and R.J.S., unpublished), which was crossed
with the floxed COUP-TFII mice. As lacZ is only turned on in
cells with COUP-TFII locus recombined or deleted, X-gal staining can
be used as a marker for successful COUP-TFII recombined or deleted
cells. As shown in Fig. 2B,
X-gal staining of an E12.5 stomach from
Nkx3-2Cre/+; COUP-TFIIflox/+ embryo
demonstrates that COUP-TFII was ablated specifically in the gastric
mesenchyme (Fig. 2C). This is
expected because X-gal staining of lacZ knock-in mice is detected
only in the stomach primordium (Fig.
2A) which is similar to Nkx3-2-Cre expression at E9.5, as
demonstrated by the product of intercrossing the Nkx3-2Cre
with ROSA26 reporter strain (Fig.
2B).

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Fig. 2. Ablation of COUP-TFII in the mesenchyme of the developing stomach
using Nkx3-2Cre and COUP-TFII floxed mice. (A)
Whole-mount X-gal staining of Nkx3-2Cre/+;
COUP-TFIIflox/+ embryo, demonstrating the ablation of
COUP-TFII by Nkx3-2Cre at E9.5 is shown. lacZ expression
represents Cre-mediated recombination of floxed COUP-TFII.
(B) The expression of Nkx3-2Cre at E9.5 was detected by whole-mount X-gal
staining of Nkx3-2Cre/+; ROSA26R/+ embryo. X-gal
staining represents Cre-mediated ablation of an interfering stop sequence in
the ROSA26 reporter gene. Specific staining was observed in the
developing stomach of both embryos. (C) A partially dissected
Nkx3-2Cre/+;
COUP-TFIIflox/+ embryo was stained by whole-mount X-gal
staining, then paraffin embedded and serially sectioned. X-gal staining
indicates Cre-mediated recombination and was detected throughout the gastric
mesenchyme, but not in the epithelium, demonstrating the ablation of
COUP-TFII in the gastric mesenchyme at E12.5. Scale bars: in B, 100
µm for A,B; in C, 100 µm for C.
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Conditional ablation of COUP-TFII in the gastric mesenchyme results in dysmorphogenesis and radial patterning defects of the developing stomach
The size of the mutant stomach is slightly smaller when compared with the
control littermate at E12.5. Histological analysis of Hematoxylin and
Eosin-stained sagittal sections of the mutant stomach showed that the
epithelium is considerably thicker than the controls at E12.5
(Fig. 3A,B). In addition, the
circular smooth layer, which is stained with
-smooth muscle actin
antibody, is disorganized in comparison with the controls at E13.5
(Fig. 3C,D). These abnormal
morphological changes were again seen at E14.5 (data not shown), suggesting
radial patterning of the stomach might be perturbed.

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Fig. 3. Expansion of smooth muscle layers and enteric motoneurons in conditional
mutant stomach. (A-D) Embryos were obtained by mating with COUP-TFII
floxed homozygous males and Nkx3-2Cre/+;
COUP-TFIIflox/+ females. COUP-TFII floxed
homozygote served as controls, and Nkx3-2Cre/+;
COUP-TFIIflox/flox were designated as conditional mutants.
Histological analysis of stomach dissected from E12.5 controls (A) and
conditional mutants (B) showed that the epithelium of the conditional mutant
is thicker (marked by the black arrows) in Hematoxylin and Eosin stained
sagittal sections. At E13.5, the smooth muscle layers of the mutant stomach
are disorganized in comparison with the controls, as seen by -smooth
muscle actin immunostaining (brown) (compare C with D). (E-H) -smooth
muscle actin staining of sagittal sections of E16.5 stomach. Smooth muscle
cells were immunoassayed for -smooth muscle actin (green), and
counterstained with propidium iodide (red). White arrows indicate the
extension of -smooth muscle actin staining in the submucosal mesenchyme
of the conditional mutant (F). (E,G,H) The thickened circular smooth muscle
layer formation was observed in both the fore-stomach (F) and the hind-stomach
(H) of the conditional mutant in comparison with the control (E,G). (I,J)
Semi-thin section semi-thin sections of the stomachs from E15.5 embryos were
examined. The circular smooth muscle layer of the conditional mutant stomach
(J) is disorganized in comparison with the control (I). (K,L) PGP9.5 staining
of E16.5 stomach. Enteric neurons were stained by protein gene product 9.5
(PGP9.5) antiserum (brown) and counterstained with Hematoxylin. (M,N) TUJ1
staining of E13.5 stomach. Anti-TUJ1 antibody was employed in immunostaining.
An increase of TUJ1-positive cells is shown in the conditional mutant (N) in
comparison with the littermate control (M).
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To further assess the potential radial patterning defects exhibited by the
conditional COUP-TFII-null mutants, we examined the differentiation
of smooth muscle and enteric neurons of the mutant stomach at later
development. Immunostaining of
-smooth muscle actin confirmed the
presence of a thickened circular smooth muscle layer formation in both the
fore-stomach (Fig. 3E,F) and
hind-stomach of conditional mutant mice
(Fig. 3G,H). Moreover,
submucosal extension of
-smooth muscle actin positive cells in the
mutant fore-stomach (Fig. 3F, white arrows) indicated precocious differentiation of smooth muscle cells. To
further demonstrate that the smooth muscle layer in the conditional mutant
stomach is disorganized, semi-thin sections
(Fig. 3I,J) and the
ultrastructure (data not shown) of the stomachs from E15.5 embryos were
examined. Again, circular smooth muscle layer of the conditional mutant
stomach is disorganized (Fig.
3J) in comparison with the controls
(Fig. 3I) at E15.5 as revealed
by semi-thin sections. However, cellular defects, other than the
disorganization of cell layers, have not been observed (data not shown).
Immunostaining for PGP9.5, an enteric neuron marker, showed an increased
number of positively stained cells in the conditional mutant mice
(Fig. 3L). To ensure there is
expansion of enteric neurons in the mutant stomach, an additional neuronal
marker, TUJ1 (neuronal class III tubulin-ß) was employed in
immunostaining. An increase of TUJ1-positive cells is shown in the conditional
mutant (Fig. 3N) in comparison
with the littermate control (Fig.
3M). Taken together, conditional ablation of COUP-TFII in
the mesenchyme resulted in at least two patterning abnormalities, epithelium
outgrowth and expansion of circular smooth muscle and enteric neuron, both
radial-patterning defects of the stomach. The fact that COUP-TFII is only
deleted in the mesenchyme compartment but the epithelium of the mutant stomach
is expanded, suggesting signals from the mesenchyme is essential for proper
growth of the epithelium.
Anteroposterior patterning of the stomach is altered in the COUP-TFII conditional null mutant
The abnormal radial patterning of the stomach manifested by the
COUP-TFII mutants prompted us to assess if other patterning defects
were also present in the developing mutant stomach. A whole-mount view
revealed dysmorphogenesis in the stomach of conditional mutants
(Fig. 4A,B). The anatomical
demarcation between the stratifying fore-stomach epithelium and the columnar
hind-stomach epithelium, referred to as `the limiting ridge' as indicated by
broken line, begins to develop, forming a presumptive margin between the
fore-stomach and hind-stomach. The size of the fore-stomach compartment and of
the entire organ was noticeably reduced in the conditional mutants
(Fig. 4C,D, white arrowheads
indicate the junction of fore- and hind-stomach). Detailed histological
examination reveals abnormalities in both the epithelium and mesenchyme. The
glandular epithelium was thicker in mutants when compared with controls
(Fig. 4E-H). In control mice,
the epithelium was thickest at the pyloric region (to the left of
Fig. 4E), and the thickness of
the epithelium progressively decreased anteriorly (towards the right). By
contrast, the epithelium of conditional mutant mice remained thick even in
more anterior regions (Fig.
4F). The mesenchyme of the conditional mutant mice was also
thickened. The hind-stomach epithelium showed several invaginations in the
control mice (Fig. 4G), while
more extensive invagination associated with the thickened epithelial layer in
the conditional mutant mice (Fig.
4H). The morphological reduction of the fore-stomach and the
extension of the hind-stomach suggest aberrant compartmentation of the
stomach, a possible patterning defect in the anteroposterior axis.

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Fig. 4. Analysis of AP patterning defects in conditional COUP-TFII mutant
stomach. Controls and conditional mutant embryos were obtained by mating with
COUP-TFII floxed homozygous males and
Nkx3-2Cre/+; COUP-TFIIflox/+ females
as described in Fig. 3. (A,B)
Stomachs were dissected from E16.5 embryos and examined in whole mount under
the dissecting microscope. The presumptive margin between fore-stomach and
hind-stomach is indicated by dots and is shifted slightly anteriorly in the
mutant. (C,D) Whole-mount postnatal day 28 stomachs were dissected and
examined under the dissecting microscope. Position of limiting ridge is
indicated by arrowhead. A clear anterior shift of the limited ridge is
observed in the conditional mutant. (E,F) Dissected stomachs from E16.5
embryos were longitudinally sectioned and stained with Hematoxylin and Eosin.
(G-H) Higher magnification of similar regions as indicated by lines in E,F. e,
epithelium; sm, smooth muscle layer; fs, fore-stomach; hs, hind-stomach; oe,
esophagus; d, duodenum.
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Histological analysis of whole embryo sagittal sections were carried out to
examine if indeed patterning of anteroposterior axis in the conditional mutant
is perturbed, Analysis of E16.5 conditional mutants revealed an alteration in
epithelial characteristics along the AP axis
(Fig. 5A-H). The proximal
duodenum exhibited typical characteristics for this stage in both control
(Fig. 5A) and conditional
mutant mice (Fig. 5E). However,
the pylorus of conditional mutant mice exhibited a disorganized thickened
circular smooth muscle layer, with an epithelium that resembled the epithelium
of the duodenum and was devoid of vacuoles
(Fig. 5F), unlike the highly
vacuolated control (Fig. 5B).
The hind-stomach of controls had a simple columnar epithelium with few
invaginations, and a tight circular smooth muscle layer
(Fig. 5C). By contrast, the
hind-stomach of conditional mutant mice exhibited a vacuolated epithelium
(Fig. 5G), resembling the
pyloric region of control mice (Fig.
5B), with numerous invaginations and a thickened but disorganized
circular smooth muscle layer. In addition, a cell layer located distal to the
circular smooth muscle layer (Fig.
5G, indicated by small arrows at the bottom), where enteric
neurons are normally located, was expanded in the conditional mutants.
Finally, the fore-stomach epithelium begins to stratify at E16.5, with
characteristic rough and deep infolding that was evident in the control mice
(Fig. 5D). The conditional
mutant mice showed similar infolding, but epithelium did not show clear signs
of stratification (Fig. 5H). In
fact, the fore-stomach epithelium of conditional mutant mice more closely
resembled hind-stomach epithelium (Fig.
5C). These observable morphological changes in the
compartmentation of different regions of the stomach are consistent with the
notion that the stomach is posteriorized.

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Fig. 5. Morphological changes in the posteriorized conditional mutant stomach:
E16.5 whole embryos were sectioned sagittally and stained with Hematoxylin and
Eosin for histological assessment. Corresponding regions in mutant and control
were estimated by morphological characteristics of mesenchyme of each regions
and relative location against other anatomical structures. (A,E) Duodenum.
(B,F) Pyloric region. (C,G) Hind stomach. (D,H) Fore stomach. Oblique arrows
across control and conditional mutant panels indicate changes of epithelial
characteristics in the conditional mutants. e, epithelium; sm, smooth muscle
layer.
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Alteration of AP patterning in the fore-stomach
If AP patterning of the stomach is indeed disrupted in the conditional
mutant, it is anticipated that the specification/differentiation of gastric
epithelium might be affected. To assess if the fore-stomach is altered, we
examined the fore-stomach regions using the stratified epithelial cell marker
cytokeratin 14 (CK14). Immunostaining using CK14-specific antibody showed
intense staining in the E16.5 control fore-stomach and the very anterior
region of the mutant fore-stomach (data not shown). CK14 staining remained as
intense throughout the fore-stomach of the control
(Fig. 6A). However, it was
barely detectable in the more caudal regions of the presumptive fore-stomach
of the mutant (Fig. 6B),
suggesting that the mutant stomach is posteriorized in which only the very
anterior region of the stomach epithelium is stratified and expresses CK14,
while the hind-stomach is morphologically expanded and is non-stratified.

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Fig. 6. Marker gene analyses confirm morphological changes in the conditional
mutant stomach. (A,B) E16.5 sagittal sections were stained for stratified
epithelial cell marker CK14. Intense staining was found in the basal cells in
the control (A), while only faint staining was observed in the anterior edge
of fore-stomach of the COUP-TFII conditional mutant (B). (C,D) Whole
E11.5 embryos were cross-sectioned and Shh expressions in the
fore-stomach were examined by section in situ hybridization using
35S-UTP labeled probes. Positive signals were pseudo-colored (red)
and overlaid on bright-field image of Hematoxylin staining. (E-G) Paraffin
sections of E18.5 stomachs were stained with anti-CK14 antibody. CK14 staining
(dark brown) remained as intense throughout the fore-stomach of the control
(E) and mutant (F). Again, it was barely detectable in the more caudal regions
of the stomach of the mutant (G) in comparison with similar anatomical
position of fore-stomach of the control (E).
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Shh has been shown to be highly expressed in the fore-stomach and can serve
as a fore-stomach marker. In the controls, Shh is highly expressed in the
fore-stomach and the expression extends into the hind-stomach
(Fig. 6C). By contrast, the
expression of Shh is anteriorly restricted in the conditional mutant
(Fig. 6D). The anterior
restriction of Shh expression observed in the conditional mutants is
similar to that detected in the stomachs of Hoxa5 mutant, indicating
posteriorization of the fore-stomach (Aubin
et al., 2002
).
Similar result for CK14 staining was observed at the later stage of
development. CK14 staining remained as intense throughout the fore-stomach of
the control and mutant at E18.5 (Fig.
6E,F). Again, it was barely detectable in the more caudal regions
of the stomach of the mutant (Fig.
6G), in comparison with similar anatomical position of
fore-stomach of the control (Fig.
6E). This result further support the notion that the mutant
stomach is posteriorized in which only the very anterior region of the stomach
epithelium is stratified and expresses CK14, whereas the hind-stomach is
expanded and is non-stratified.
The expression of the differentiated hind-stomach markers remains unchanged in the conditional mutants
As shown earlier, the radial patterning and the AP patterning of the
stomach are altered in the mutants at E13.5, leading to the respective
thickening of the epithelium and the expansion of the hind-stomach (Figs
3,
4,
5). To examine if these
phenotypes persist at later development, we examined the mutant stomach at
E18.5. Similar to early development, it is clear that the epithelium is
thickened and the hind-stomach of the mutant is considerably expanded
(Fig. 7B) in comparison with
the controls (Fig. 7A). To
examine whether differentiation of the glandular epithelium is perturbed in
the mutant, the expression of parietal cell marker
H+/K+-ATPase ß-subunit and of the glandular gastric
epithelium marker GATA4 were analyzed at E18.5. Although
H+/K+-ATPase ß-subunit positive parietal cells are
present in the glandular gastric epithelium of both controls
(Fig. 7C) and conditional
mutant mice (Fig. 7D), the
number of the parietal cells were increased in the thickened epithelial layer
of the conditional mutant mice (Fig.
7D). The expression of GATA4 in the thickened glandular epithelial
layer of the mutant (Fig. 7F) is similar to that of the controls (Fig.
7E), suggesting differentiation of the glandular epithelium is
unchanged in the mutant.

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Fig. 7. (A,B) Dissected stomachs from E18.5 embryos were longitudinally sectioned
and stained with Hematoxylin and Eosin. The thickness of the glandular
epithelium is increased in the mutant stomach compared with the littermate
control. (C-F) The adjacent tissue sections between fore-stomach and
hind-stomach were stained by anti-H+/K+-ATPase
ß-subunit antibody (C,D) and anti-GATA4 (E,F) antibodies. The transition
zone between oxyntic mucosa (os) and stratified squamous epithelium (sse) of
the fore-stomach are indicated by arrows. (G-T) Marker gene analyses in the
adult stomachs: (G-L) Sections (4 µm) prepared from the stomachs of
35-day-old mice were staining with Hematoxylin and Eosin. The histological
morphology of zymogenic region (G,H), mucoparietal zone (I,J) and pure mucus
zone (K,L) showed no obvious difference in the conditional mutant stomach
(H,J,L) and littermate control (G,I,K). (M,N) The secreted glycoprotein of the
zymogenic zone of stomachs was visualized by PAS staining and no discernable
difference was observed in the conditional mutant stomach (N) and littermate
control (M). (O,P) The gastric parietal cells were stained with
anti-H+/K+-ATPase ß-subunit antibody. (Q,R) The
neck and pre-neck cells (red) at the gastric units showed no difference in the
control (Q) and conditional mutant (R) stomachs. (S,T) Anti-GATA4 antibody was
used to mark glandular gastric epithelium. Positive signals were found in base
of glandular gastric epithelium at pure mucus zone in the control (S) and the
mutant (T).
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To further substantiate that differentiation of the hind-stomach is largely
intact in the mutant, we examined the mutant hind-stomach at adulthood.
Histological analysis of the stomachs from 35-day-old of mutant mice and
littermate controls showed no obvious difference in the hind-stomachs at the
regions of Zymogenic zone (Fig.
7G,H), mucoparietal zone (Fig.
7 I,J) and pure mucus zone
(Fig. 7K,L) by Hematoxylin and
Eosin. Mucins secretion was analyzed by periodic acid-Schiff (PAS) staining
and similar PAS staining was observed in both control
(Fig. 7M) and mutant
(Fig. 7N) stomachs. To further
examine if there is any changes in cell specification in the mutant
hind-stomach, we employed H+/K+-ATPase ß-subunit as
the parietal cells marker (Fig.
7O,P), GSII as the neck and pre-neck cell marker
(Fig. 7Q,R) and GATA4 as marker
in the base of the pure mucus unit (Fig.
7S,T) in the analysis. The expression profiles of all these
markers are similar in the mutants when compared with the controls. These
results, taken together, support the notion that there are no cell type
changes in the conditional mutant in comparison with littermate control
throughout development even though there are observable patterning defects in
the stomach.
Alteration in the very caudal part of the mutant hind-stomach
As histological analysis indicated that the very caudal part of the
hind-stomach might have acquired pylorus characteristics, we used alkaline
phosphatase (EAP) to determine whether hind-stomach is indeed posteriorized.
As shown in Fig. 8A, EAP is
mainly expressed in the duodenum and pylorus region of the controls. By
contrast, high level of EAP expression not just found in the duodenum and
pylorus, but it was also found in the hind-stomach of conditional mutant mice
using longitudinally sectioned E16.5 dissected stomach
(Fig. 8B). Clear anterior
extension of EAP-positive epithelium, as indicated by brackets, was found in
the hind-stomach of conditional mutant mice. Although expression of the
markers H+/K+-ATPase ß-subunit
(Fig. 8C,D) and GATA4
(Fig. 8E,F) is no different in
the regions anterior to the junction of the pylorus and duodenum from E18.5
control (Fig. 8C,E) and mutant
(Fig. 8D,F) stomachs, the
higher level of EAP activity was again found in the mutant hind-stomach
(Fig. 8H,J). Taken together,
the extended and high levels of EAP expression in the hind-stomach again
indicate that patterning of the AP axis is abnormal in the conditional
mutants.

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Fig. 8. (A,B) Paraffin sections of E16.5 stomachs were stained for EAP activity.
The bracket indicates corresponding regions of the caudal end of control and
mutant stomach. oe, esophagus; Py, pylorus; d, duodenum. (C,D) The junction
region of the caudal end of the stomach and the duodenum show slight
morphological differences. The gastric parietal cells were stained with
anti-H+/K+-ATPase ß-subunit antibody. (E,F)
Anti-GATA4 antibody was used to mark glandular gastric epithelium. (G-J)
Paraffin sections of E18.5 stomachs were stained for EAP activity. The bracket
indicates corresponding regions of the caudal end of control and mutant
stomach. Very low positive signals were found in glandular gastric epithelium
of the control (G,I) while highly intense signals were seen in the mutant
(H,J). High magnification of the boxed corresponding regions in the control
(G) and mutant (H) were shown in I and J, respectively. Scale bar in B: 100
µm for A,B.
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Does COUP-TFII mediate Hh signaling in the stomach?
The radial and AP patterning defects elicited by the
COUP-TFII-null mutants bear resemblance to the dysmorphogenesis of
the stomach in Shh-null mutant mice
(Ramalho-Santos et al., 2000
).
Because of this similarity, we asked if COUP-TFII expression is
altered in the Shh-null mutant. Whole-mount in situ hybridization showed that
COUP-TFII expression in the caudal region of the stomach of the
Shh-null mutant is downregulated in comparison with the control
littermate. However, the expression of COUP-TFII in other regions of
the stomach is only slightly downregulated
(Fig. 9A,B). An anterior
shifting of the caudal margin of COUP-TFII expression at the
gastro-duodenal junction (indicated by broken lines; presumptive
gastro-duodenal junction is indicated by arrowheads based on morphology and
location of the dorsal mesogastrium) was evident in the Shh-null
mutant. In addition, a significant size reduction, particularly in the
fore-stomach was noted in the Shh-null mutant stomachs, even as early
as E12.5 stage, using the esophagus as a guide
(Fig. 9B, indicated by white
lines).

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Fig. 9. Effect of inhibition of Hh signaling in COUP-TFII expression.
(A,B) Developing stomachs were dissected from E12.5 Shh wild-type and
null mutant, and expression of COUP-TFII mRNA was detected by
whole-mount in situ hybridization. Caudal margins of COUP-TFII
expression are indicated by dots, and presumptive anatomical boundaries
between stomach and duodenum are indicated by arrowheads. The relative size of
fore-stomach is indicated by a white line at the top of the stomach. (C-F)
Foregut explants were dissected from E10.5 knock-in embryos and cultured for
48 hours in increasing concentrations of cyclopamine (0-10 µM, D-F). Strong
lacZ expression was detected in the mesenchyme between lung bud and
fore-stomach, as indicated by arrowhead, and cyclopamine inhibited
lacZ expression in a dose-dependent manner. Scale bars: in B, 0.5 mm
for A,B; in F, 0.5 mm for C-F. oe, esophagus; fs, fore-stomach; hs,
hind-stomach; dmg, dorsalmesogastrium; lb, lung bud; St, stomach; Py, pylorus;
d, duodenum; e, hind-stomach epithelium.
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As the presence of Ihh in the Shh-null mutant stomach
(Bitgood and McMahon, 1995
)
might functionally compensate for Shh and influence
COUP-TFII expression, we used cyclopamine to inhibit all Hh
signals (Chen et al., 2002
) and
asked if COUP-TFII expression is affected in the explants organ
culture. Explants dissected from E10.5 lacZ knock-in embryos were
cultured for 48 hours in the presence of increasing amounts of cyclopamine.
Whole-mount X-gal staining of COUP-TFII illustrated a significant
dose-dependent reduction of COUP-TFII/lacZ expression, especially in
the mesenchyme between lung bud and fore-stomach (indicated by arrowheads)
(Fig. 9C-F). At 1 µM of
cyclopamine, a slight reduction of COUP-TFII mRNA, as represented by
X-gal staining, was detected in the stomach explants from E11.5 embryos. At 5
µM and 10 µM of cyclopamine, the reduction of X-gal staining was more
pronounced in the stomach when compared with the controls
(Fig. 9E,F), while
COUP-TFII mRNA was not affected in lung explants (not shown). The
downregulation of COUP-TFII expression when Hh signaling is inhibited by
cyclopamine substantiates the notion that Hh signaling regulates
COUP-TFII expression in the stomach.
 |
Discussion
|
---|
The lacZ knock-in mice allow us to follow the expression of
COUP-TFII during gastric development. It is apparent that COUP-TFII
is expressed in both the mesenchyme and the epithelium of the developing
stomach. The graded mesenchymal expression profile, with highest levels in
mesenchyme subjacent to the epithelium, suggests a diffusible molecule
secreted by the epithelium may regulate the expression of COUP-TFII.
COUP-TFII is also expressed in the glandular epithelium of the stomach,
with highest expression in the base where the zymogenic cells are localized,
but is not expressed in the surface pit cells. The expression patterns in the
zymogenic unit resemble that of Shh (van
den Brink et al., 2001
). Taken together of the above findings, we
speculate that Shh might regulate COUP-TFII in the stomach in a
similar manner as previously shown in the motoneurons
(Krishnan et al., 1997a
).
Conditional ablation of COUP-TFII in the mesenchyme of the
developing stomach by Nkx3.2 Cre recombinase firmly established that
COUP-TFII is essential for radial patterning of the stomach. At
E12.5, it is already apparent that the circular smooth muscles layers and the
enteric neurons layers are expanded and disorganized in the conditional
mutants. These defects persist at later stages of development. Although
COUP-TFII in the epithelial compartment has not been deleted, the
epithelium is considerably thicker in comparison with the control littermates,
indicating signals from the mesenchyme are required for appropriate epithelium
growth. The defects display by the COUP-TFII conditional mutants
indicate that COUP-TFII is essential for radial patterning of the
developing stomach and these defects are not seen in the compound heterozygote
of Nkx3.2 and COUP-TFII. Instead, the inappropriate
differentiation of the smooth muscles cells and enteric neurons in the
mesenchyme has been shown when Hh signaling is disrupted by treatment with
cyclopamine (Sukegawa et al.,
2000
; van den Brink et al.,
2001
). Shh is believed to be the epithelial signal that inhibits
the gut mesenchyme to differentiate into smooth cells and the neural crest
cells to differentiate into enteric neurons. Our findings indicate that
COUP-TFII participates in the negative regulation of differentiation of smooth
muscle cells and enteric neurons in the gut mesenchyme. COUP-TFII can either
serve as a downstream target of Hh to mediate its negative function in the gut
mesenchyme or exert its effect in a pathway parallel to Hh to regulate
mesenchymal differentiation.
In addition to radial patterning of the stomach, COUP-TFII is also required
for AP patterning of the stomach. The anterior shift of the limited ridge that
divides the fore- and hind-stomach, the reduced size of the fore-stomach, the
expansion of the hind-stomach and the expansion of EAP expression into the
hind-stomach are all consistent with disruption of the AP axis patterning in
the stomach. However, the differentiation of the epithelium of the fore- and
hind-stomach in the adult mutant remains unchanged; with all the molecular
markers analyzed, even the mutant stomach is posteriorized. The
posteriorization and ectopic extended expression of EAP in the hind-stomach of
the conditional mutant have been demonstrated in the Shh-null mutant
mice (Ramalho-Santos et al.,
2000
). The striking similarity of the phenotypes exhibited by
conditional COUP-TFII-null mutants and animals, chick and mouse, when
Hh signaling is disrupted further implicates that Hh and COUP-TFII act in the
same and/or parallel pathways.
Although the general slight reduction of expression of COUP-TFII
in the Shh-null mutant is a little surprising, the high expression of
Ihh in the expanded hind-stomach of the Shh-null mutant could have
compensated for the missing Shh to induce COUP-TFII expression.
Indeed, the downregulation of COUP-TFII expression in the mesenchyme of the
stomach is more pronounced in the explants treated with cyclopamine,
supporting the notion that COUP-TFII is a downstream target of Hh
signaling. In addition, Shh signaling can directly activate COUP-TFII
expression and Shh-N activates COUP-TFII expression in P19 cells
without de novo protein synthesis
(Krishnan et al., 1997a
;
Krishnan et al., 1997b
).
Furthermore, analysis of the COUP-TFII promoter has identified an
ShhRE that is distinct from the defined Gli-binding site
(Krishnan et al., 1997a
).
Thus, all the above results indicate that COUP-TFII mediates Hh signaling in
the stomach. Taken together, our results established that COUP-TFII is
essential for radial and AP patterning of the stomach. Restriction of anterior
Shh expression and attenuation of Shh action in the
epithelium of the COUP-TFII conditional mutant, in turn, suggests a
potential role for COUP-TFII in the stimulation or maintenance of Shh
expression.
Shh derived from the epithelium signals the mesenchyme of the stomach, but
how mesenchymal factors influence endodermally expressed Shh is
unclear. The Hoxa5-null mutant phenotype provides evidence for
mesenchyme-mediated regulation of Shh expression in the developing
stomach (Aubin et al., 2002
).
Similarly in chick, it was suggested that adjacent mesenchyme regulates
Shh expression (Narita et al.,
1998
). In our study, COUP-TFII was ablated in the
mesenchyme and the observed dysmorphogenesis suggests that COUP-TFII
potentially affects such mesenchymal factor(s). One such candidate,
Bmp4, which belongs to the TGFß superfamily, is expressed in the
mesenchyme of developing murine stomach, while the chick ortholog has been
demonstrated to play a role in gizzard patterning. Bmp4 is important
for mesoderm development, but because of early lethality in Bmp4-null
mutants, no specific role for Bmp4 in gastric development has been
elucidated (Winnier et al.,
1995
). Interestingly, it has been shown that COUP-TFII
binds to the mouse Bmp4 promoter and regulates Bmp4 promoter
activity in transient transfection assays
(Feng et al., 1995
), suggesting
COUP-TFII might function through BMP signaling. Another member of TGFß
super family, activin, governs embryonic axial patterning, and restricts
Shh expression in Hensen's node
(Monsoro-Burq and Le Douarin,
2001
). Furthermore, the expansion of Shh expression
domain within the stomach of Acvr2a/Acvr2b mutant mice indicates a
restriction of Shh in the foregut that is potentially regulated by
TGFß/Bmp signaling. As Acvr2 is able to interact with BMPs
(Kim et al., 2000
),
mesenchymal expressed Bmp4 may restrict Shh expression.
Alternatively, it is possible that COUP-TFII may modulate the expression of
activin/activin receptor themselves. Enhanced activin signaling may restrict
the expression domain of Shh to the anterior border and perturb the epithelial
growth, as seen in the conditional mutant stomach. Further analysis of
TGFß/Bmp/activin signaling in the COUP-TFII conditional mutant
stomach may shed light on which mesenchymal factor(s) that regulate
Shh expression are affected by the absence of COUP-TFII.
 |
ACKNOWLEDGMENTS
|
---|
We thank Xiaoyan Huang, Kikue Takamoto and Wei Qian for excellent technical
help; Dr Milton Finegold, Mr James Barrish and the Core Morphology laboratory
of the NIH Gulf Coast Digestive Disease Center for the thin-section and the EM
analysis; Drs Zijian Lan, Hong Wu and Dennis Roop for various suggestion and
discussion; Drs Kimi Araki, Takeshi Yagi and Matthew Scott for plasmid DNA;
and Kurt Schillinger, Debra Bramblett and Peter Tsai for preparation of
manuscript. NIH grants DK55636 to S.Y.T. and DK45641 to M.J.T. supported this
work.
 |
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