Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, 701 W 168th Street, New York, NY 10032, USA
* Author for correspondence (e-mail: vep1{at}columbia.edu)
Accepted 3 February 2003
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SUMMARY |
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Key words: T-box, Yolk sac, Limb development, Tbx3, Ulnar-mammary syndrome, UMS, Mammary gland
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INTRODUCTION |
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TBX3 and the mouse ortholog, Tbx3, which was discovered
by Bollag et al. (Bollag et al.,
1994), are members of the Tbx2 subfamily of the T-box family of
transcription factor genes. The family is characterized by a highly conserved
sequence encoding a DNA-binding domain, called the T-domain, that binds
specific sequences in the promoters of target genes. The Tbx2 subfamily, which
includes Tbx2, Tbx3, Tbx4 and Tbx5, evolved by tandem
duplication of an ancestral locus to form a linked pair of genes, followed by
duplication of the gene pair and dispersal to two chromosomal locations. In
both mouse and human, Tbx2 and Tbx4 are linked, and
Tbx3 and Tbx5 are linked
(Agulnik et al., 1996
;
Papaioannou, 2001
). The
relatively recent separation of the paralogs, Tbx2 and Tbx3,
is reflected in
90% identity in amino acid sequence in the T-box and
similarities in expression patterns and function of the two genes
(Agulnik et al., 1996
;
Brummelkamp et al., 2002
;
Chapman et al., 1996b
;
Gibson-Brown et al., 1998a
;
Gibson-Brown et al., 1998b
;
Jacobs et al., 2000
;
Lingbeek et al., 2002
). Both
of the mouse genes and their human counterparts can act as transcriptional
repressors (Carlson et al.,
2001
; Carreira et al.,
1998
; He et al.,
1999
), and although no downstream target genes have yet been
identified for mouse Tbx3, the human genes TBX3 and
TBX2 were both identified in functional screens as negative
regulators of the cell cycle control gene Cdkn2a. TBX2 and
TBX3 repress the Cdkn2a transcript
p14ARF in humans (known as p19ARF in
mouse) through an interaction with a variant T-domain binding site that is
specific to these two T-box genes
(Brummelkamp et al., 2002
;
Jacobs et al., 2000
;
Lingbeek et al., 2002
).
Recently, the crystal structure of TBX3 in complex with DNA has been reported
and suggests that TBX3 binds as a monomer to its natural target sites
(Coll et al., 2002
).
The highly specific patterns of T-box gene expression during development
implicate them in many developmental processes
(Papaioannou, 1997;
Papaioannou, 2001
;
Smith, 1999
). In addition to
the UMS mutations in TBX3, mutations in several other T-box genes
confirm their vital developmental roles in a variety of metazoan species,
including human and mouse (Basson et al.,
1997
; Bruneau et al.,
2001
; Chapman et al.,
1996a
; Jerome and Papaioannou,
2001
; Russ et al.,
2000
). In general, the phenotypic abnormalities observed in these
mutants correlate with areas of gene expression. In mouse, Tbx3 is
first expressed in the inner cell mass (ICM) of the blastocyst, and later in
the mesoderm and endoderm layers of the yolk sac, and in the mesoderm of the
amnion, chorion and allantois during gastrulation. During organogenesis,
Tbx3 is expressed in the primordia of many tissues and organs,
notably in the apical ectodermal ridge (AER) and the anterior and posterior
margins of the developing limbs, and in the epithelium of the mammary buds
(Chapman et al., 1996b
;
Gibson-Brown et al., 1996
;
Gibson-Brown et al., 1998b
;
Yamada et al., 2000
).
Tbx3 is also expressed in various adult organs
(Bollag et al., 1994
). Less is
known about the spatial pattern of expression in humans, but the gene is
expressed in a variety of adult organs, as well as in fetal lung, kidney,
heart, liver, spleen and pituitary gland
(Bamshad et al., 1999
;
Bamshad et al., 1997
). It has
been postulated that the abnormalities seen in individuals with UMS result
from a haploinsufficiency of TBX3 that leads to hypoplasia of tissues
through an interference with apoptosis or differentiation during critical
stages of embryogenesis (Bamshad et al.,
1997
; Carlson et al.,
2002
). To provide a mouse model of UMS in order to understand the
developmental role of Tbx3, we have generated a mutation in mouse
Tbx3 by gene targeting in embryonic stem cells. In contrast to human
UMS mutations, the mouse Tbx3 mutation is recessive on several
genetic backgrounds, with the exception of a mild defect in female genitalia.
However, the homozygous mutant phenotype reflects the predominant features of
UMS with severe defects in forelimb and mammary gland development and, in
addition, reveals a novel role for Tbx3 in the yolk sac.
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MATERIALS AND METHODS |
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In situ hybridization, immunohistochemistry and histology
Whole-mount in situ hybridization was performed as described previously
(Wilkinson, 1992). PECAM-1
antibody staining (PharMingen; San Diego, CA) on whole yolk sacs was performed
using standard protocols (Davis et al.,
1991
). Apoptosis was examined in yolk sacs from 9.5 dpc and 10.5
dpc embryos that were living at the time of recovery (two mutant and two
wild-type at each age) using immunohistochemistry for activated caspase (Cell
Signaling technology, NEB, Beverley, MA)
(Di Cunto et al., 2000
). For
detection of mammary bud induction in homozygous mutant embryos, embryos alive
at the time of recovery were examined by in situ hybridization for
Wnt10b (11.5 and 12.5 dpc) and Lef1 (12.5 and 13.5 dpc),
along with stage and age-matched controls. Embryos (12.5 and 13.5 dpc) were
examined histologically in serial sections as described above for evidence of
mammary bud development. The following markers of limb development were
examined by in situ hybridization on mutants and age/stage matched controls:
Shh (10.5, 11.5 and 12.5 dpc); Fgf8, engrailed 1
(En1), Wnt7a and Tbx2 (10.5 and 11.5 dpc);
Msx1, Tbx4 and Tbx5 (10.5 dpc); Hand2 (9.5 and 10.5
dpc).
Adult mammary gland analysis
Inguinal mammary glands were collected from six-week-old virgin females,
pregnant females (12.5 days of gestation), lactating females (2 weeks after
parturition) and postlactation females (1 week after weaning). The virgin and
pregnant females were from both 129 inbred and C57BL/6Tac-129 mixed background
mice (F2 and F2 intercross), whereas the lactating and
postlactation glands were from the mixed background only. Three or four glands
were examined from each genotype and background. In each case, the right
inguinal mammary gland (number 4) was isolated from the skin by blunt
dissection and scraping with a scalpel, and then spread onto a glass slide for
overnight fixation in Carnoy's fixative. Fixed glands were stained in Carmine
Alum, using standard procedures (Kordon
and Smith, 1998). Glands were scored using a semi-quantitative
measure for the extent of ductal development and the ratio of terminal end
buds to end buds.
Skeletal preparations and measurements
Standard methods were used for Alcian Blue/Alizarin Red skeletal
preparations of embryos (Mallo and
Brändlin, 1997). For measurements of the posterior zeugopod
bones, late fetal stage embryos (17.5 dpc for ulna measurements and 18.5 dpc
for fibula measurements) were stained and the limb bones were measured using a
grid under a dissecting microscope.
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RESULTS |
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Embryonic lethality and yolk sac defects in Tbx3 homozygous
mutant mice
No Tbx3tm1Pa homozygous mutants were recovered at term
in 18 litters from inter se matings of Tbx3tm1Pa/+ mice.
Examination of embryos during gestation revealed that homozygous mutants were
present at the expected Mendelian ratios
(Table 2A;
2=0.0318, P>0.05), but about half of them (17/35)
were dead by 11.5 dpc and none survived beyond about 16.5 dpc
(Table 2B). The occasional
abnormalities seen in +/+ or Tbx3tm1Pa/+ embryos ranged
from open brains to a placenta with no embryonic remains, whereas the
phenotype of Tbx3tm1Pa/Tbx3tm1Pa
homozygous mutant embryos consists of three major abnormalities: yolk sac
defects, lack of mammary glands and limb defects. Prior to the onset of
lethality at 10.5 dpc, some homozygous mutant embryos have normal yolk sacs
(Fig. 3E,F), but others (6/24)
show a reduction in yolk sac vasculature
(Fig. 3A). In these mutant
embryos, yolk sac blood vessels were small or absent, as confirmed by
immunostaining for an endothelial marker, PECAM
(Fig. 3B-D), and sometimes
contained no detectable blood, although blood was present in the heart and
embryonic circulation. At 9.5 and 10.5 dpc, no difference in the amount of
apoptosis was detected between mutant and wild-type yolk sacs of living
embryos using activated caspase immunohistochemistry (data not shown). By 12.5
dpc, however, most mutant embryos were dead (21/33) and the yolk sacs of many
of the surviving mutants were abnormal. Histologically, the endoderm layer of
abnormal-appearing yolk sacs consisted mostly of dead cells with pyknotic
nuclei, and the blood vessels had begun to deteriorate
(Fig. 3G,H). No obvious
abnormalities were seen in placental morphology or morphology of fetal organs
and tissues with the exception of a smaller than normal liver (see
Fig. 2M-P). No surviving
homozygous mutant embryos were observed beyond 13.5 dpc, although two dead
fetuses were recovered near term that had survived to about 16 dpc, judging
from their developmental stage (Fig.
4E-H).
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Limb abnormalities in
Tbx3tm1Pa/Tbx3tm1Pa embryos
Homozygous mutant embryos are normal at 8.5 dpc, but about 70% of mutants
are identifiable by reduced hindlimb bud development by 9.5-10.5 dpc, whereas
the forelimb bud appears to be normal in size and shape at this stage. By 11.5
dpc, all homozygous embryos that were alive, or were dead but sufficiently
intact to be scored, displayed both fore- and hindlimb abnormalities, with
irregularities in the hand plate and either very little development or an
irregular shape to the foot plate that resembled a posterior deflection of the
autopod (Table 2B;
Fig. 4A-D). The AER is present
in both fore- and hindlimbs, although it appears reduced in the forelimb and
more severely reduced in the hindlimb as indicated by Fgf8 expression
(Fig. 5A). Msx1, which
marks cells in the progress zone, was expressed in a normal graded pattern in
the fore- and hindlimbs of Tbx3 homozygous mutant embryos, although
the expression in the forelimb appeared to be restricted posteriorly (data not
shown). Dorsoventral patterning of the limb is normal, as indicated by the
expression of Wnt7a in the dorsal ectoderm and engrailed 1 in the
ventral ectoderm (data not shown). The expression of two T-box genes
implicated in the specification of fore-versus hindlimb, Tbx5 and
Tbx4, respectively, is normal
(Fig. 5C,D). However,
Tbx2, a closely related T-box gene with normal expression largely
overlapping that of Tbx3 in the limb bud margins, shows reduced
expression in the posterior margin of the forelimb and no expression in the
posterior margin of the hindlimb of Tbx3 homozygous mutants
(Fig. 5B). Sonic hedgehog
(Shh) expression, which marks the zone of polarizing activity (ZPA)
at the posterior limb margin, was present in fore- and hindlimbs of three out
of four wild-type embryos at 10.5 dpc, and in both limbs of all wild-type
embryos at 11.5 dpc. In mutant embryos, however, there is no expression in
stage-matched mutants at 10.5 dpc (Fig.
5E) and only one out of four mutants showed any Shh
expression at 11.5 dpc. In this embryo, expression was reduced in the
forelimbs and limited to a very small area in the hindlimbs
(Fig. 5F). Hand2,
which is initially expressed in the flank and then becomes localized to the
posterior limb buds (Charite et al.,
2000), is normally expressed in the flank and posterior forelimb
bud at 9.5 dpc in mutant and wild-type embryos. However, by 10.5, mutant
embryos (n=4) showed a reduced domain of posterior forelimb bud
expression compared with stage-matched controls, and none has expression in
the hindlimb buds (Fig.
5G).These early limb abnormalities are reflected in corresponding
abnormalities of the limb skeleton of the small number of homozygous mutants
that reach late fetal stages. In one fetus of
16.5 dpc
(Fig. 4E-G), the right forelimb
had a shortened ulna and was missing the metacarpals and phalanges of the
fifth digit, and phalanges of the fourth digit. The left forelimb lacked an
ulna and carpals as well as metacarpals and phalanges of the fourth and fifth
digits (Fig. 4G). In the more
severely affected hindlimb, the femur was present, but a single misshapen
element represented the zeugopod. A single digit was attached to the zeugopod
element of the right hindlimb. In a second fetus of the same approximate age,
the right forelimb was shortened but contained an ulna, radius and five
digits. The left forelimb was missing the ulna, and the metacarpals and
phalanges of the fourth and fifth digits. In the hindlimbs, the femur was
present bilaterally; the left hindlimb resembled that of the fetus described
above, with a single digit attached to the zeugopodal element; the right
hindlimb had a femur, a tibia and a single digit attached to the tibia
(Fig. 4H). The pelvic bones
were small and poorly formed in both fetuses
(Fig. 4G).
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DISCUSSION |
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Several possibilities could explain these species differences: they could
be the result of differences in genetic background modifiers, species-specific
differences in dose sensitivity between mice and humans, or alternatively,
differences in the developmental role of Tbx3 between mouse and
human. With respect to genetic background effects, humans are generally
considered an outbred population and could be segregating for genetic
modifiers, which might explain the variable phenotype seen in different
individuals with identical TBX3 mutations. However, in mice with
uniform genetic backgrounds (inbred mice and F1 mice), we have
observed variable expressivity. We have also observed a similar variability
and range of expressivity in several mixed genetic backgrounds, indicating
that phenotypic variability is not due solely to genetic background
differences but must also be caused by stochastic events during development.
Species differences in dose sensitivity between humans and mice have been
observed for a number of other genes [e.g. SHH
(Chiang et al., 1996;
Ming et al., 1998
;
Roessler et al., 1996
) and
PAX9 (Peters et al.,
1998
; Stockton et al.,
2000
)], including another T-box gene, TBX1, which
produces a severe haploinsufficient phenotype in humans that is reflected in a
mild and transient heterozygous phenotype in mice
(Jerome and Papaioannou, 2001
;
Lindsay et al., 2001
;
Merscher et al., 2001
).
Finally, although it appears that the human mutation is completely penetrant
in the pedigrees studied, it could be that other families harbor mutations
that go undetected in the heterozygous condition.
The difference in severity between the forelimb and hindlimb is somewhat
harder to explain and could represent functional differences in the
developmental role of Tbx3 in limb growth and patterning, or species
differences in the expression of Tbx3 as have been observed between
chick and mouse (Gibson-Brown et al.,
1998a). Tbx2 and Tbx3 are normally expressed in
virtually identical patterns in the margins of early limb buds. It is possible
that these closely related genes have partially overlapping functions, and
that human and mouse have evolved a different balance of function between the
two. As yet, no mutations in Tbx2 have been reported in either
species to test this idea. Notwithstanding these species differences, the
major aspects of the Tbx3 mutant phenotype in limb, genitalia and
mammary gland in mice correspond to abnormalities characteristic of UMS, and,
in addition, a yolk sac defect not reported in humans has been uncovered.
Deficiency of mammary gland induction in Tbx3 mutant
mice
The mammary gland hypoplasia seen in individuals with UMS is reflected in
the deficiency in mammary placode induction in homozygous mutant mice and the
absence or reduction of mammary buds in mutant embryos. Although there appears
to be no haploinsufficiency effect on mammary development or function in
heterozygotes, in Tbx3 homozygous mutant mice, a severe deficiency of
mammary development is seen at the earliest stages of mammary bud induction,
as indicated by the absence of the two earliest markers of the mammary
placode, Wnt10b and Lef1, and lack of morphological evidence
of placodes. Normally, five pairs of mammary placodes form between 11 and 12.5
dpc, but in 13 Tbx3 homozygous mutants examined either histologically
or with molecular probes between 11.5 and 13.5 dpc, only three buds were
detected, all of them histologically: one 13.5 dpc embryo had a single, small
mammary bud and a second 13.5 dpc embryo had a pair of buds. These embryos
were smaller than their normal littermates, but were at comparable
developmental stages, as judged by developmental landmarks such as the
development of the pinnae of the external ear.
Studies using targeted gene mutations have begun to yield information on
the earliest stages of mammary placode induction, and several signaling
pathways have been implicated in its control. The five mammary placodes are
induced asynchronously between 11 and 12.5 dpc with placode 3 appearing first
and placode 2 being the last to form
(Mailleux et al., 2002). In
addition, induction of the inguinal bud (placode 4) is different from the
others in that it is independent of the FGF10/FGFR2 pathway, although its
later maintenance depends on FGFR2 activity. Mice with a null mutation of
either Fgf10 or Fgfr2 develop only the inguinal bud, which
is maintained in the absence of FGF10 but not in the absence of FGFR2
(Mailleux et al., 2002
).
Induction of this inguinal bud also appears to be independent of WNT
signaling, as mice lacking LEF1, an effector of WNT/ß-catenin signaling,
also develop only a single pair of inguinal mammary buds
(van Genderen et al., 1994
).
Mice that lack Msx2 expression show variable arrest of mammary gland
development, but Msx1, Msx2 double homozygotes reveal redundancy for
these two transcription factors in early mammary gland induction; placodes
form but fail to undergo further development into mammary bud
(Satokata et al., 2000
),
although the status of each individual placode was not reported in this study.
The deficiency of mammary placodes in mice lacking Tbx3 argues that
Tbx3 is required for mammary bud induction and acts upstream of both
the FGF and WNT signaling pathway because all of the placodes, including the
FGF- and WNT-signaling independent placode 4, are affected. It may be that
Tbx3 is required for mammary epithelium to become competent for
continued development, although the rare, late appearance of placode 2
indicates that this requirement is not absolute.
Limb abnormalities in Tbx3 mutant mice
The deficiency in the development of posterior limb elements in individuals
with UMS is reflected in limb abnormalities in mutant mice, including
deficiency of the forelimb digits and ulna, as well as the foot and fibula,
that result from a failure in the development of posterior limb elements. Limb
development and patterning depends on reciprocal interactions between the AER
and the ZPA, involving complex signaling through the FGF and SHH signaling
pathways (Martin, 2001). FGF
signaling from the limb mesenchyme is essential for the formation of the AER,
which then induces the ZPA in the posterior part of the limb. Shh
signaling from the ZPA is primarily responsible for anteroposterior (AP)
patterning of the limb and is regulated by a complex feedback loop with FGFs
in the AER (Sun et al., 2000
).
Tbx3 is normally expressed both in the AER and in the limb mesenchyme
at the anterior and posterior margins of the limbs
(Chapman et al., 1996b
;
Gibson-Brown et al., 1996
);
therefore, it could be involved in the initiation or maintenance of Fgf gene
expression in the AER, and/or Shh expression in the ZPA.
The abnormalities in anteroposterior patterning seen in Tbx3
homozygous mutant limbs closely resemble the limb phenotype seen in mice with
homozygous mutations in Shh, or with a limb-specific reduction of
Shh expression (Chiang et al.,
2001; Kraus et al.,
2001
; Lewis et al.,
2001
), suggesting that Tbx3 normally functions in the SHH
pathway, and that loss or reduction of Shh is responsible for the
limb phenotype in Tbx3 mutants. In both Tbx3 and
Shh mutants, the posterior tissue of the fore- and hindlimbs is
reduced and the AER appears weak and disorganized, as shown by FGF8
expression. In addition, only digit one, a Shh-independent digit,
forms in the hindlimb of Tbx3 homozygous mutants.
The similarities in the limb abnormalities observed in Tbx3 and
Shh mutant embryos combined with the absence or reduction of
Shh expression in Tbx3 homozygous mutants suggest a role for
Tbx3 in the initiation and/or maintenance of Shh expression.
Tbx3 is not absolutely required for Shh expression because a
small proportion of Tbx3 homozygous mutant embryos express
Shh, as shown by the presence of Shh in one out of four
mutants at 11.5 dpc, and by the normally patterned right forelimb in one late
stage mutant fetus. We propose that Tbx3 functions with
Hand2 during early limb patterning to regulate Shh
expression and set up the initial anteroposterior patterning of the limb.
Shh expression is then required to maintain and expand the early
anteroposterior pattern, and to maintain Hand2 and Tbx3
expression in the posterior limb, as the posterior domain of Tbx3
expression has been shown to be Shh dependent
(Tumpel et al., 2002).
Alternatively, or in addition to this role in regional signaling in limb
patterning, Tbx3 may play a role in maintaining proliferation in the
posterior limb margin. Both Tbx3 and the closely related
Tbx2 have recently been identified in senescence bypass screens to
identify genes that allow the bypass of proliferation arrest
(Brummelkamp et al., 2002;
Jacobs et al., 2000
),
implicating these genes in the maintenance of cellular proliferation. A
deficiency in the number of posterior limb margin cells in mutants could
result in a reduced ZPA, with fewer cells producing SHH. The feedback loop
maintaining the AER would consequently be affected. The posterior reduction in
Msx1 expression, which is an AER-independent marker of the progress
zone, would fit with a reduction in posterior tissue. Whether the lower
expression of Tbx2 in the posterior margins of the limbs of mutant
mice is due to loss of posterior tissue or an interaction between
Tbx2 and Tbx3 has yet to be determined. This decrease in
posterior Tbx2 expression could also be a direct result of decreased
Shh expression, as it has been shown that Shh can regulate
Tbx2 (Gibson-Brown et al.,
1998a
).
Embryonic lethality of Tbx3 homozygous mutants
No homozygous TBX3 humans have been reported. Our mouse model
provides a tentative explanation for this in that the yolk sac, which is vital
for hematopoiesis and maternal-fetal exchange during gestation, is severely
compromised in mutant mice. Although the yolk sac vasculature initially forms
normally in the majority of mutant embryos and blood is produced, massive cell
death of the yolk sac endoderm occurs at a variable time during gestation,
even though Tbx3 expression in the yolk sac and other fetal membranes
appears to be transitory in the early stages of yolk sac development
(Chapman et al., 1996b). In
the absence of the detection of other life-threatening abnormalities in fetal
membranes or in the fetus, and because some embryos still have a beating heart
at the same time as the yolk sac defects are evident, we attribute embryonic
lethality to the yolk sac failure, which includes, but is not limited to the
vasculature. If there is a similar role for TBX3 in human fetuses,
they too would be lost during gestation. At present, we have no explanation
for the variable time of death or the variable expressivity of the yolk sac
phenotype, and it is not clear what developmental pathways are affected. It is
possible that some mutant embryos survive with a threshold level of yolk sac
development only to succumb to delayed effects of an earlier deficiency in the
metabolic function of the yolk sac or deficiencies in the vasculature, blood
islands or hematopoietic precursors. The small size of the liver in mutant
embryos might be a reflection of this deficiency.
The putative function of Tbx3 in the p19ARF/Mdm2/p53
pathway (Brummelkamp et al.,
2002; Carlson et al.,
2002
; Jacobs et al.,
2000
; Lingbeek et al.,
2002
) offers a possible explanation for the cell death observed in
the yolk sac, as well as the hypoplasia of the mammary bud and posterior limb
elements. The failure of Tbx3 to suppress p19ARF
expression in these areas of Tbx3 embryonic expression could lead to
the upregulation of this gene and the subsequent upregulation of p53.
This in turn could lead to cessation of proliferation and/or increased
apoptosis in the yolk sac and eventual death of the fetus. This possibility is
under investigation.
In summary, the homozygous Tbx3 mutant mouse recapitulates two of the major features of UMS, the posterior limb defect and mammary gland hypoplasia, as well as uncovering an additional role for the gene in the yolk sac. Tbx3 mutant mice provide a means of investigating the involvement of this gene in specific signaling pathways during development of the yolk sac, limb and mammary gland.
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ACKNOWLEDGMENTS |
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