1 Kennedy Krieger Research Institute, Baltimore, MD 21205, USA
2 Department of Neuroscience, Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA
3 Department of Biological Chemistry, Johns Hopkins University School of
Medicine, Baltimore, MD 21205, USA
4 Department of Dermatology, Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA
* Author for correspondence (e-mail: thompsonc{at}kennedykrieger.org)
Accepted 9 June 2004
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SUMMARY |
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Key words: Hair follicle, Alopecia, Nuclear receptor, Repression, Epidermis, hr
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Introduction |
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The mouse mutant hairless (Hr; previously known as
hr) was first recognized in 1926 for its characteristic hair loss
phenotype, in which initial hair growth is normal but after shedding the hair
does not grow back (Brooke,
1926). The Hr gene was identified by mapping the
retroviral insertion in the original murine allele
(Cachon-Gonzalez et al., 1994
;
Stoye et al., 1988
) and as a
thyroid hormone-regulated gene in rat brain
(Thompson, 1996
).
Identification of the human ortholog revealed that mutations in the
Hr gene result in congenital hair loss disorders (alopecia
universalis, papular atrichia) (Ahmad et
al., 1998b
; Cichon et al.,
1998
; Sprecher et al.,
1999
). Multiple mutant Hr alleles in both mice and humans
show phenotypic variations that can include skin wrinkling and papular rash
(Panteleyev et al., 1998b
).
Although mechanisms have been proposed to explain histological changes in
Hr mutant skin that include absence of hair follicles, epidermal
utricles and dermal cysts (Mann,
1971
; Montagna et al.,
1952
; Panteleyev et al.,
1999
), the precise role of Hr in hair follicle biology
remains unknown.
The Hr gene encodes a 130 kDa protein (HR) that is highly
expressed in skin and brain
(Cachon-Gonzalez et al., 1994;
Potter et al., 2001a
).
Although the HR protein lacks sequence identity to proteins of known structure
or function, we recently demonstrated that HR functions as a nuclear receptor
corepressor (Potter et al.,
2001a
). Nuclear receptors are transcription factors that regulate
specific changes in gene expression in response to the binding of their
cognate ligands (Mangelsdorf et al.,
1995
; McKenna and O'Malley,
2002
). The transcriptional activity of nuclear receptors depends
on the association of additional proteins (co-activators and co-repressors),
many of which function in chromatin remodeling
(Glass and Rosenfeld, 2000
;
Jepsen and Rosenfeld, 2002
).
HR interacts with and influences the transcriptional activity of multiple
nuclear receptors, including thyroid hormone receptor (TR), retinoic acid
receptor-related orphan receptor
(ROR
) and vitamin D receptor
(VDR) (Hsieh et al., 2003
;
Moraitis et al., 2002
;
Potter et al., 2001a
;
Thompson and Bottcher, 1997
).
In the context of TR, HR mediates repression in the absence of thyroid
hormone, probably through interaction with histone deacetylases
(Potter et al., 2001a
;
Potter et al., 2002
). In the
case of ROR
, a constitutively active orphan receptor, HR inhibits
transcriptional activation via a novel mechanism using co-activator-type
binding motifs (LXXLL) (Moraitis et al.,
2002
). HR function in the context of VDR is distinct, as HR also
inhibits transcriptional activation by the ligand-bound receptor
(Hsieh et al., 2003
).
The nuclear receptors with which HR interacts have been implicated in
epithelial development and function. In humans, thyroid hormone (TH)
deficiency frequently causes thickening of the skin and hair loss
(Bernhard et al., 1996;
Freinkel and Freinkel, 1972
).
Like mutations in Hr, some mutations in the gene encoding VDR result
in congenital hair loss in mice and humans
(Malloy et al., 1999
;
Miller et al., 2001
).
Similarly, conditional inactivation in mouse skin of the genes encoding
heterodimeric partners of TR and VDR (RXR
and RXRß results in
progressive alopecia (Li et al.,
2001
; Li et al.,
2000
). The phenotypic similarities of Hr, VDR and RXR
(retinoid X receptor) mutant animals suggest that the biochemical interaction
of these proteins is functionally relevant in vivo.
As HR is a transcriptional regulator, the phenotype of Hr mutants likely results from a perturbation of gene expression. To study the role of HR in vivo, we generated a null allele (Hr-) using homologous recombination. In this study, we find specific changes in gene expression in Hr-/- skin, which includes upregulation of keratinocyte differentiation markers. Based on the properties of the cell types present in Hr-/- skin, we propose that the role of HR in the skin is to regulate the timing of epithelial progenitor cell differentiation, and that disruption of timing in Hr mutants leads to changes in cell fate favoring epidermis and sebaceous glands at the expense of the hair follicle.
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Materials and methods |
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RNA and protein analysis
Mouse strains C57BL/6, HRS/J Hr, SKH2/J and RHJ/LeJ
Hrrh-J were obtained from The Jackson Laboratory;
CBA-Hrrh was obtained from Taconic. For RNA preparation,
mouse backskin was pooled from two male and two female mice for each age and
genotype, and RNA was isolated using an RNeasy kit (Qiagen). Primary
keratinocytes were cultured in EMEM (0.05mM Ca2+) with 8% chelex
treated fetal bovine serum as described
(Filvaroff et al., 1994;
Hennings et al., 1980
;
Yuspa et al., 1989
). Cultures
were treated with 0.12 mM Ca2+ for 24 hours before harvesting total
RNA using Trizol (Sigma).
Microarray analysis was performed using Affymetrix gene array chips (U74Av2) following Affymetrix specifications (Johns Hopkins Medical Institutions Core Facility). First-strand cDNA was synthesized using oligonucleotide probes with 24 oligo-dT plus T7 promoter as primer (SuperScript Choice System, Life Technologies). After synthesis of double-stranded cDNA, biotinylated antisense cRNA was generated by in vitro transcription using the BioArray RNA High Yield Transcript Labeling kit (ENZO Life Sciences). Biotinylated cRNA was fragmented and hybridized to the GeneChip array, followed by two rounds of staining with a streptavidinphycoerythrin conjugate. Image analysis was carried out using the Agilent GeneArray Scanner and MicroArray Suite 5.0 software (Affymetrix). For comparison between different chips, global scaling was used, scaling all probe sets to a user defined target intensity of 150. Significant changes were defined as greater than twofold with a P value of less than 7x10-6.
Northern analysis was as described
(Potter et al., 2001b).
Results were quantitated using a Fujifilm BAS-2500 phosphorimager to scan two
independent blots for each probe; values were normalized to signal for
ß-tubulin. DNA fragments used as probes were from the indicated plasmids:
rat Hr cDNA (Thompson,
1996
); keratin 10 (pETK10); loricrin (ATCC clone 1747977);
filaggrin (ATCC clone 949741); caspase 14 (ATCC clone 3451156); Kdap
(ATCC clone 1477125); calmodulin 4 (ATCC clone 1766225) and ß-tubulin (A.
Lanahan, Dartmouth University).
For protein preparation, mouse backskin was frozen, crushed and homogenized
in 1.5 ml of an isotonic detergent buffer. After centrifugation, 15-20 µl
of supernatant was loaded onto a SDS-polyacrylamide gel. Western analysis was
performed as described (Potter et al.,
2001a).
Generation of Hr-/- mice
The targeting vector was constructed from the plasmid PGK-tk by first
subcloning the PGK NEO loxP cassette from pKSneo-12 (C.-M. Fan, Carnegie
Institution of Washington, Baltimore, MD). PCR amplification of 129SV genomic
DNA with specific primers was used to generate fragments spanning exon 6-7 (2
kb) and exon 11-17 (4.5 kb). Fragments were inserted into restriction sites
flanking the NEO cassette. The targeting vector was linearized and
electroporated into 129/Sv embryonic stem (ES) cells (JHU Transgenic Core
Facility), followed by selection with G418 and gancyclovir. Colonies were
screened using Southern analysis with probes flanking the recombination site
(Fig. 1, data not shown). Four
out of 143 colonies showed homologous recombination. Chimeric mice were
generated using two independent ES cell lines. C57BL/6J mice were mated with
chimeric mice to generate heterozygous animals. Offspring were genotyped by
Southern analysis of EcoRI-digested genomic DNA. Subsequent
generations were genotyped using PCR. Heterozygotes were interbred to obtain
Hr-/- animals. Studies were carried out with mice of mixed
background; preliminary studies with seventh generation backcross to C57BL/6
have yielded comparable results.
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Results |
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To generate a defined mouse model that lacks Hr expression, a
targeted deletion of the Hr locus was created using homologous
recombination in mouse embryonic stem (ES) cells
(Fig. 1C). The targeting
construct was designed to excise exons 8-10, to both disrupt the 3 kb
Hr mRNA (which initiates in exon 8) and remove a functional domain
required for interaction with nuclear receptors
(Moraitis et al., 2002;
Potter et al., 2001a
).
Chimeric mice were generated from ES cells carrying the recombined allele and
crossed to C57BL/6 mice to produce heterozygous animals. Heterozygotes were
intercrossed to generate mice with two copies of the recombinant allele, which
segregated in a Mendelian fashion. Mice homozygous for the Hr
targeted gene deletion were identified by the presence of the recombinant
allele (6 kb) and absence of the wild-type allele (9 kb) using Southern
analysis (Fig. 1D). Although
the recombinant allele allows for the potential production of truncated RNA
and protein (exons 1-6), no full-length Hr mRNA was detected
(Fig. 1E). In addition, no
full-length or truncated HR protein was detected in the skin of mice carrying
the recombinant allele using an antibody that recognizes epitopes that would
be present in the putative truncated protein
(Fig. 1F). We refer to the
mouse strain homozygous for the targeted allele as
Hr-/-.
Examination of Hr-/- mice during postnatal development
shows that, as in other Hr alleles, the first hair coat grows
normally. Hair loss is observed at P18, beginning at the head and
proceeding caudally in a scattered pattern. Once the hair is lost, the skin of
Hr-/- mice appears wrinkled, similar to that of rhino
alleles. The histology of Hr-/- skin is also initially
similar to previously described alleles as the pilary canal widens to form a
utricle, hair follicles fail to regenerate and small dermal cysts become
visible. However, as the Hr-/- mice age, the skin becomes
progressively more wrinkled (Fig.
1G). When compared with Hrhr/Hrhr
and Hrrh/Hrrh mutants,
Hr-/- mice exhibit a more prominent wrinkling phenotype
(Fig. 1H). This wrinkling
phenotype resembles that of the rhino-Yurlovo allele, which (like
Hr-/-) is caused by an insertion that would disrupt both
Hr mRNAs (Ahmad et al.,
1998c
; Cachon-Gonzalez et al.,
1994
; Panteleyev et al.,
1998a
), therefore the severe wrinkling phenotype is probably due
to complete loss of Hr expression.
Utricle formation in Hr-/- skin
Utricle formation is the first morphological change observed in
Hr-/- skin. Histological analysis pointed to postnatal day
12 as the age at which a change is visible, near the top of the pilary canal
(infundibulum) (Fig. 2A). Once
formed, the utricle remains a prominent feature as the hair is lost and
persists as hair follicles fail to regenerate. The mechanism of utricle
formation is unclear. To characterize the cells that comprise the utricle,
immunohistochemical detection differentiation of keratinocyte markers was
examined at P19 (Fig. 2B).
Keratin 10 (K10), which localizes to the suprabasal layer of the epidermis in
wild type skin, was detected both in the epidermis and in the utricle walls in
Hr-/- skin. Keratin 14 (K14) is normally expressed in
basal cells of the epidermis and pilosebaceous unit. In
Hr-/- skin, K14 was present in the basal layer of the
epidermis as well as the basal layer surrounding the utricle and sebaceous
gland. Keratin 17 (K17), which localizes to the outer root sheath (ORS) in
wild-type epidermis, was expressed in cells that comprise the utricle walls.
Filaggrin, a marker of late stage epithelial differentiation, was detected in
the innermost layers of the utricle wall (data not shown, see
Fig. 7). In contrast to
epidermal markers, the utricle does not express a hair marker (trichohyalin)
(Fig. 2C). Both K14 and K17
were detected in cell clusters in the dermis distal to the utricles and
sebaceous glands; these cells may represent remnants of the receding hair
follicle.
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The expansion of the utricle led us to examine cell proliferation. Identification of mitotically active cells at P16 (prior to hair loss) showed that the number of proliferating cells in the epidermis is greater in Hr-/- than in wild-type skin (Fig. 3A). In Hr-/- skin, proliferation above wild-type levels is observed both in the utricle and the interfollicular epidermis. Cells in the utricle region remain mitotically active as hair follicles undergo telogen (Fig. 3B) and after hair loss (data not shown). Mitotic activity in the infundibulum prior to hair loss may contribute to utricle formation and continuing proliferation may help maintain the utricle. Detection of keratin markers in combination with BrdU incorporation indicated that proliferating cells are K14-positive (Fig. 3B). Keratin 16 (K16) expression remains comparable in wild-type and Hr-/- skin, suggesting that the infundibulum is not a site of aberrant differentiation (Fig. 3C). Thus, the increase in cell proliferation in Hr-/- skin is probably a direct consequence of loss of HR function. As the animals age, increased proliferation probably contributes to the increase in skin surface area, and is also necessary to accommodate tissue expansion that results from cyst formation.
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We next assessed which epithelial compartment contributes to the increase in mRNA of the most highly regulated genes, caspase 14 and filaggrin (Fig. 7). At P6, caspase 14 and filaggrin are expressed in the infundibulum and the suprabasal layer of the epidermis in both wild-type and Hr-/- skin. At this age, caspase-14 and filaggrin expression in Hr-/- skin is similar in abundance and expression pattern to wild-type skin, consistent with northern analysis. As postnatal development proceeds, expression of both genes is downregulated in the interfollicular epidermis in both wild-type and Hr-/- mice. As Hr-/- skin matures (P9-P19), caspase 14 and filaggrin expression in the infundibulum not only persists but increases as the utricle develops. Increased gene expression occurs before the morphologically distinct utricle can be identified, indicating that the upregulation is probably a cause rather than a consequence of utricle formation. Indeed, filaggrin expression in Hr-/- skin (P9) is detected in the shape of a utricle despite the absence of a histologically detectable utricle structure. Expression of other identified upregulated genes was also detected in utricles (data not shown).
The observed increase in gene expression, prior to utricle formation, suggests that transcription of such genes is normally inhibited by HR. To determine whether expression of the upregulated genes is normally repressed by HR, we examined expression in primary newborn keratinocytes cultured from Hr-/- and Hr+/- mice. Using northern analysis, we find that expression of both filaggrin (2.9-fold) and caspase 14 (2.0-fold) is significantly higher in Hr-/- keratinocytes grown in elevated Ca2+ (0.12 mM) to promote differentiation (Fig. 8) and in proliferating keratinocytes grown in low Ca2+ (data not shown). Thus, the increase in filaggrin and caspase 14 expression in Hr-/- skin is not simply a result of utricle formation, and probably occurs because HR represses transcription of these genes.
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Discussion |
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Similarly, work over the past 50 years examining histology, cell
proliferation and cell death in spontaneous Hr alleles led to
predictions that HR coordinates the balance between cell proliferation,
differentiation and/or apoptosis in the epidermis and hair follicle
(Mann, 1971;
Montagna et al., 1952
;
Panteleyev et al., 1999
). By
combining detailed molecular analysis of the cell types present in
Hr-/- skin with current models of epithelial cell fate
determination, our results provide evidence that HR has a role in regulating
the timing of cell differentiation in the skin. HR plays a similar role in
both the epidermis and the hair follicle, in both cases influencing cell
fate.
Gene expression is altered in Hr-/- skin
Our recent demonstration that the HR protein functions as a nuclear
receptor co-repressor suggested that HR regulates gene expression through its
interaction with other proteins. We show here that loss of HR function results
in specific changes in gene expression. The identified genes showed increased
expression in Hr-/- skin relative to wild type, consistent
with removing the function of a transcriptional repressor. Temporally, we
detect changes in gene expression well before the onset of the Hr
mutant phenotype. Spatially, we find that altered gene expression in
Hr-/- skin is first detected in the infundibulum and is
restricted to epidermal structures (utricle). The change in expression is
intrinsic to keratinocytes, as expression is upregulated in keratinocytes
isolated from Hr-/- skin, suggesting that HR directly
represses transcription of the identified genes. As both phenotypic
alterations and significant changes in gene expression in Hr mutant
skin occur in the infundibulum, the utricle is probably derived from the
infundibulum by a combination of changes in gene expression and altered cell
proliferation.
Many of the misregulated genes are involved in keratinocyte terminal
differentiation (K10, loricrin, filaggrin), consistent with the role of HR in
the epidermis. The roles of other upregulated genes are less obvious, but
their expression and putative functions suggest that they too have a role in
keratinocyte terminal differentiation. The most highly upregulated gene,
caspase 14, is a member of the caspase family of proteins and is the only
caspase with tissue-restricted expression
(Ahmad et al., 1998a;
Eckhart et al., 2000a
;
Hu et al., 1998
;
Van de Craen et al., 1998
).
Although caspase 14 does not cleave classical caspase substrates and is not
activated by apoptosis-inducing agents, the protein is processed during
epidermal differentiation and processing is associated with terminal
keratinocyte differentiation (Chien et
al., 2002
; Eckhart et al.,
2000b
; Lippens et al.,
2000
). Calmodulin 4/Scarf is a Ca2+-binding
protein expressed exclusively in differentiating keratinocytes, and is
proposed to control Ca2+-mediated signaling in epidermal
differentiation (Hwang and Morasso,
2003
; Koshizuka et al.,
2001
). Kdap was isolated based on its specific expression
in developing epidermis, and is expressed in suprabasal cells of epidermis and
the infundibulum (Oomizu et al.,
2000
). Thus, although the functions of caspase 14, calmodulin
4/Scarf and Kdap are unclear, these genes probably have
important roles in the skin, and their aberrant expression may promote the
conversion of infundibulum to epidermis.
HR action as a co-repressor fits well with the role of transcriptional
regulatory proteins in mediating hair and skin development and function. As a
co-repressor, HR does not directly regulate transcriptional activity but
instead acts in concert with other transcription factors. Both biochemical and
physiological evidence supports the idea that in the skin, HR acts at least in
part through VDR: RXR heterodimers (Hsieh
et al., 2003). Physiologically, the phenotypes of Hr, VDR
or RXR
mutants are similar as initial hair growth is normal but
subsequent hair cycles fail (Li et al.,
2001
; Li et al.,
2000
; Li et al.,
1997
; Miller et al.,
2001
; Yoshizawa et al.,
1997
). In addition, VDR mutants show reduced epidermal
differentiation, indicating that VDR also has a role in epidermis
(Xie et al., 2002
). However,
the phenotypes are not identical as RXR
mutants show a
hyperproliferative response in the epidermis
(Li et al., 2001
). In
addition, hair loss in VDR and RXR mutants is delayed relative to Hr,
and neither show severe wrinkling. These phenotypic variations indicate that
HR may also act through other nuclear receptors in the skin, such as TR, and
may influence the activity of other transcription factors as well.
Role of HR in regulating cell differentiation
The Hr mutant phenotype is observed in temporally and spatially
distinct compartments, the epidermis and hair follicle. We propose that the
role of HR in both compartments can be understood within the context of
epithelial cell differentiation. Continuous regeneration of the epidermis and
cyclical regeneration of the hair follicle both rely on a pool of epithelial
stem cells that reside in specialized parts of the outer root sheath (ORS)
(Braun et al., 2003;
Cotsarelis et al., 1990
;
Fuchs et al., 2001
;
Oshima et al., 2001
). Stem
cells give rise to multipotent progenitor cells that migrate in a
bidirectional manner (Taylor et al.,
2000
). Cells that travel upwards ultimately populate the epidermis
and sebaceous glands, while cells that migrate downwards normally contribute
to the regenerating hair follicle. Progenitor cells differentiate into
distinct cell fates by responding to cues provided by multiple signaling
molecules, which include secreted factors such as WNTs, SHH and BMP4, and
transcription factors such as MYC, LEF1 and TCF3
(Kratochwil et al., 1996
;
Merrill et al., 2001
;
Millar et al., 1999
;
Niemann et al., 2002
;
St-Jacques et al., 1998
;
Waikel et al., 2001
;
Wilson et al., 1994
).
Our data support a model in which the multiple phenotypic changes in
Hr mutant skin result from the loss of a repressive influence on
differentiation. Normally, multipotent progenitors originating from the bulge
and/or other locations within the ORS transit through the infundibulum on
their way to the epidermis (Ghazizadeh and
Taichman, 2001; Kratochwil et
al., 1996
; Niemann and Watt,
2002
). Keratinocytes located in the infundibulum exhibit a
relatively high proliferation rate, but do not terminally differentiate
(Morris et al., 2000
;
Schmitt et al., 1996
). In the
absence of HR, infundibular keratinocytes continue to proliferate; however,
differentiation is no longer inhibited. Instead, these cells adopt an
epidermal fate in response to local cues. This interpretation is supported by
Hr mRNA expression in the infundibulum of mature hair follicles, and
by the conversion of the infundibulum to utricles in Hr-/-
follicles.
The idea that HR regulates entry into differentiation can also account for the phenotypic alterations seen in the lower part of the hair follicle. Multipotent progenitor cells migrating downward from the ORS-associated stem cells should normally give rise to all the lineages required to form a hair bulb capable of producing hair. In the absence of HR, the inductive signal(s) required to trigger the stem cells into forming a hair bulb are missing, and in response to signals from the prevailing microenvironment, they instead attempt to produce sebaceous glands. This results in the absence of hair follicles and development of an abnormal dermal structure, the cyst. Continuing cell proliferation leads to expansion of the cysts and skin, probably contributing to the progressive wrinkling phenotype. Evidence that cysts are related to sebaceous glands includes expression of K14 and SCD1, and lack of filaggrin or K10 expression.
Precedence for the transformation of cell fates in the skin comes from
studies in which specific signaling pathways have been disrupted. For example,
inhibition of WNT signaling by inhibiting either ß-catenin or LEF1 causes
transformation of cells fated to become inner root sheath or hair into
sebocytes, thereby suppressing differentiation of hair cells and increasing
sebocyte and dermal cyst formation
(Huelsken et al., 2001;
Merrill et al., 2001
;
Niemann et al., 2002
).
Conversely, stimulation of WNT signaling by expressing a constitutive
ß-catenin results in hair follicle morphogenesis and differentiation in
cells derived from epidermis and ORS (Gat
et al., 1998
).
Although Hr is expressed early in development, phenotypic
manifestations in Hr-/- mutant skin do not appear until
the onset of postnatal hair cycling. One possibility is that loss of HR
function is effectively compensated for only during development.
Alternatively, the influence of HR on terminal differentiation is not crucial
during development, possibly because the environmental cues responsible for
the adoption of specific lineages are better segregated at spatial and
temporal levels. There are many instances in which gene manipulation
differentially affects developing skin epithelia and postnatal hair cycling
(Gat et al., 1998;
Koch et al., 1997
;
McGowan et al., 2002
).
Our results provide a unifying model for the role of HR in epidermis and hair follicle, in which HR acts alongside local environmental cues to regulate the entry of multipotent progenitor keratinocytes into specific programs of terminal differentiation. The role of HR in influencing cell differentiation can probably be extended to other tissues in which HR is expressed, such as the brain. In addition, we provide the first direct evidence that the HR protein is a transcriptional repressor in vivo, supporting a model in which the Hr mutant phenotype arises from changes in the normal pattern of gene expression that regulates the timing of epithelial cell differentiation.
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ACKNOWLEDGMENTS |
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