1 Department of Dermatology, University of Pennsylvania Medical School,
Philadelphia, PA 19104, USA
2 Department of Cell and Developmental Biology, University of Pennsylvania
Medical School, Philadelphia, PA 19104, USA
3 Center for Childhood Communication, Abramson Research Center, The Children's
Hospital of Philadelphia, Philadelphia, PA 19104, USA
4 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163, 67404 Illkirch
Cedex, France
5 Departments of Molecular, Cell and Developmental Biology, Orthopaedic Surgery,
and Biological Chemistry, David Geffen School of Medicine at UCLA, Los
Angeles, CA 90095, USA
6 Laboratory of Reproductive and Developmental Toxicology, National Institute of
Environmental Health Sciences, Research Triangle Park, NC 27709, USA
* Author for correspondence (e-mail: millars{at}mail.med.upenn.edu)
Accepted 5 February 2004
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SUMMARY |
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Key words: Hair follicle, Skin, Tooth, BMP, BMPR1A, BMPR1B, ß-catenin, Gata3, Msx, Foxn1
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Introduction |
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Periods of hair growth are followed by a regression phase (catagen), when
proliferation and differentiation of follicular epithelial cells cease and the
lower two-thirds of the follicular epithelium degenerates
(Lindner et al., 1997).
Following catagen the follicles enter a quiescent phase (telogen). The onset
of a new anagen phase is thought to involve signaling between the dermal
component of the hair follicle, the dermal papilla and epithelial cells
(Cotsarelis et al., 1990
;
Oliver and Jahoda, 1988
).
Intercellular communication between the dermis and surface epithelium, and
within the surface epithelium, is also crucial for the embryonic morphogenesis
of hair follicles and other epithelial appendages, such as teeth and mammary
glands, and several classes of secreted signaling molecules are implicated at
early stages of appendage development (reviewed by
Hardy, 1992;
Jernvall and Thesleff, 2000
;
Millar, 2002
;
Veltmaat et al., 2003
). Among
these are BMPs, which function by binding type 1 (BMPR1A and BMPR1B) and type
2 (BMPR2) transmembrane serine/threonine kinase receptors, resulting in
phosphorylation of the intracellular proteins SMADs 1, 5 and 8. Phosphorylated
SMADs bind to SMAD4 and translocate to the nucleus, where they complex with
transcription factors and regulate target gene transcription
(Mishina, 2003
). Ectopic
expression experiments and analysis of mice lacking the BMP inhibitor noggin
suggest that BMP signals act during embryogenesis to retard hair follicle
development, and may control the spacing of hair and feather follicles
(Botchkarev et al., 1999
;
Jung et al., 1998
;
Noramly and Morgan, 1998
).
Postnatally, ectopic expression studies suggest that BMP inhibition controls
anagen onset (Botchkarev et al.,
2001
), and that BMP signaling regulates hair-shaft differentiation
(Kulessa et al., 2000
).
Existing evidence suggests that BMPs play crucial roles at multiple stages of
tooth morphogenesis (Jernvall and
Thesleff, 2000
), and BMP mRNAs are also expressed in developing
mammary glands (Phippard et al.,
1996
).
Despite abundant data suggesting key roles for BMP signals in appendage
development and in postnatal hair follicle differentiation and cycling,
definitive genetic evidence from loss-of-function mutations in BMP or BMP
receptor genes is lacking because of the early lethality of most of these
mutants (Mishina, 2003). As
the BMPR1A receptor is broadly expressed in the epidermis and hair follicles
(Botchkarev, 2003
), we
investigated the requirement for epithelial Bmpr1a in these processes
using several different mouse lines that express Cre recombinase
(Lewandoski, 2001
) in
epidermal cells and their derivatives, in combination with a conditional
allele of Bmpr1a (Ahn et al.,
2001
; Mishina et al.,
2002
). Tooth morphogenesis was arrested in the most severely
affected conditional mutant mice, and postnatal development of hair follicles
was strikingly defective. Hair follicle matrix cells failed to differentiate
towards hair shaft or IRS, and expression of several known transcriptional
regulators of hair shaft and IRS differentiation was decreased or absent.
Mutant follicles failed to undergo catagen, instead continuing to proliferate
to produce matricomas and follicular cysts. These results demonstrate that
epithelial Bmpr1a plays essential roles in controlling
differentiation and proliferation in postnatal hair follicles, as well as
being required for completion of tooth morphogenesis.
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Materials and methods |
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Mice homozygous for a floxed allele of Bmpr1a
(Bmpr1afloxP-neo) (Ahn
et al., 2001; Mishina et al.,
2002
), referred to here as Bmpr1acl, were
crossed to K14-Cre or K14-Cre-ERT2
(Indra et al., 2000
)
transgenic mice, and progeny heterozygous for both K14-Cre or
K14-Cre-ERT2 and Bmpr1acl were crossed
to Bmpr1acl homozygotes. Brn4-Cre line
bcre-32 transgenic mice (Ahn et
al., 2001
) were used in combination with
Bmpr1acl and heterozygous Bmpr1a null
(Bmpr1anull) (Mishina
et al., 1995
) mice. Bmpr1anull/+ males
homozygous for bcre-32 were crossed with females of genotype
Bmpr1acl/cl. In addition, we used mice carrying a null
allele of Bmpr1b (Bmpr1bnull)
(Yi et al., 2001
):
Bmpr1anull/+; Bmpr1bnull/+ males
homozygous for bcre-32 were crossed with
Bmpr1acl/cl; Bmpr1bnull/+ females. PCR
detection of wild-type, floxed and recombined Bmpr1a alleles was as
described previously (Mishina et al.,
2002
). Newborn epidermis was isolated mechanically after
incubation of skin overnight in 0.25% trypsin in PBS at 4°C. For tamoxifen
induction, dorsal hair (if present) was clipped and 0.5 mg tamoxifen (Sigma)
in 100 µl ethanol was applied to dorsal skin three times at 48 hour
intervals. Controls were treated with vehicle only.
Histology, follicle density measurements, immunofluorescence, BrdU incorporation, TUNEL assays and in situ hybridization
Tissue samples were fixed in 4% paraformaldehyde, paraffin embedded and
sectioned at 5 µm. Samples containing bone were decalcified with 4% formic
acid, 4% hydrochloric acid for 4 hours after fixation. For histological
analysis, sections were stained with Hematoxylin and Eosin. For hair follicle
density measurements, 2-4 control animals and 2-4 experimental animals were
used for each developmental stage. Follicles were counted at 10x
magnification in 10 microscopic fields for each sample. Statistical
significance was calculated using a two-tailed Student's t-test.
Alkaline phosphatase staining for identification of dermal papillae was as
described previously (Handjiski et al.,
1994). For immunofluorescence staining, sections were microwave
pretreated and incubated with primary antibodies against phospho-SMAD1,
phospho-SMAD5 and phospho-SMAD8 (polyclonal rabbit serum, Cell Signaling
Technology; 1:50), type I hair keratin (mouse monoclonal antibody clone AE13)
(Lynch et al., 1986
) (1:10),
trichohyalin (mouse monoclonal antibody clone AE15; 1:6)
(O'Guin et al., 1992
), mouse
keratin 6 (polyclonal rabbit serum, Covance; 1:300), GATA3 (mouse monoclonal
antibody, Santa Cruz HCG3-31; 1:100) and cytokeratin K17 (polyclonal rabbit
serum; 1:500) (McGowan and Coulombe,
1998
). For detection of nuclear ß-catenin, antibody clone
15B8 (Sigma; 1:1000) was used in combination with the MOM kit (Vector
Laboratories). Polyclonal rabbit serum
(ten Dijke et al., 1994
)
(1:100) was used for detection of BMPR1A in 8 µm cryosections. Sections
were counterstained with 2 µg/ml Hoechst 33258 (Sigma).
For proliferation assays, mice were injected with BrdU (50 µg BrdU/g
body weight) and sacrificed after 1 hour. Detection of BrdU incorporation with
anti-BrdU antibody (Roche) was as previously described
(Andl et al., 2002). The In
Situ Cell Death Detection Kit (Roche Diagnostics) was used for apoptosis
assays.
In situ hybridization with 35S-labeled probes, and whole-mount
and paraffin section in situ hybridization with digoxigenin-labeled probes
were as described previously (Decimo et
al., 1995; Grachtchouk et al.,
2003
; Reddy et al.,
2001
). The following PCR products containing a T7 promoter were
used as templates for sense and antisense probe synthesis: Foxn1
(Accession Number NM_008238, nucleotides 1720-2339), and Cre
(Accession Number AF169416, nucleotides 2533-3151). Msx, Lef1, Wnt10b
and Shh probes were as described previously
(Andl et al., 2002
;
Jowett et al., 1993
).
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Results |
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Bmpr1acl heterozygotes carrying Cre
transgenes, and Bmpr1acl/cl mice lacking a Cre
transgene, were indistinguishable from non-transgenic mice that were wild type
for Bmpr1a. Bmpr1acl homozygotes carrying K14-Cre
transgenes displayed phenotypes of graded severity depending on the
Cre line used. This observation suggested subtle differences in the
level of activity of Cre recombinase in the three lines, as all of the mice
were maintained on a similar, mixed strain background. K14-Cre43;
Bmpr1acl/cl mice died within 24 hours of birth, and
displayed severe limb defects and open eyes
(Fig. 3A). The limb defects
reflected the known requirement of epithelial Bmpr1a for maintenance
of the apical ectodermal ridge (Ahn et al.,
2001). K14-Cre52; Bmpr1acl/cl mice
usually died by P4. They had grossly normal limbs and eyes, were severely
runted, and lacked external hair and teeth
(Fig. 3C,D,E).
K14-Cre40; Bmpr1acl/cl mice showed the weakest
phenotype of the constitutive K14-Cre lines and occasionally survived
for several months. These mice were runted, and showed greatly decreased
external hair (Fig. 3F-H).
K14-Cre-ERT2; Bmpr1acl/cl mice that
had not been treated with tamoxifen survived for at least 6 months. They
displayed a sparse hair phenotype that became more severe with age
(Fig. 3I). Surviving
K14-Cre40; Bmpr1acl/cl and
K14-Cre-ERT2; Bmpr1acl/cl mice had
abnormal growths under their nails (Fig.
3J). Topical tamoxifen treatment of
K14-Cre-ERT2; Bmpr1acl/cl mice caused
these animals to become sickly, requiring euthanasia 7 days after cessation of
treatment. This may have been due to trans-cutaneous absorption of tamoxifen
and induction of Bmpr1acl recombination in internal
stratified epithelia. Homozygous bcre-32 transgenic mice that were
also heterozygous for a null allele of Bmpr1a
(Bmpr1anull) (Mishina
et al., 1995
) were crossed to Bmpr1acl/cl mice
to produce progeny of genotype bcre-32;
Bmpr1acl/null, as well as bcre-32;
Bmpr1acl/+ controls. bcre-32;
Bmpr1acl/null mice survived for up to 17 days and showed
absence of external hair in the mid-ventrum, corresponding to the region of
Cre activity in ventral epidermis (Fig.
3B). The lethality of bcre-32;
Bmpr1acl/null mice was due to hydrocephalus and CNS
defects (data not shown). K14-Cre43; Bmpr1acl/cl
and K14-Cre52; Bmpr1acl/cl mice had oral
abnormalities, resulting from defective tooth development (see below), and had
difficulty suckling. Decreased lifespan of K14-Cre40;
Bmpr1acl/cl mice may have been due to loss of
Bmpr1a in internal epithelia.
|
Accelerated hair follicle morphogenesis in epithelial Bmpr1a mutants
Development of primary hair follicles in mouse embryos is initiated at
approximately E14, and secondary hair follicles develop in several waves
between approximately E16 and birth. To determine whether deletion of
epithelial Bmpr1a affects hair follicle morphogenesis, we examined
the histology of K14-Cre; Bmpr1acl/cl dorsal and
ventral skin, and bcre-32; Bmpr1acl/null
mid-ventral skin, between E16.5 and birth. In addition, anti-K17 antibody was
used to clearly reveal the location of developing hair follicles
(Fig. 4C,D). For all the
mutants analyzed, newborn skin appeared similar to littermate controls
(Fig. 4E-H and data not shown).
Measurements of follicle density in newborn dorsal K14-Cre43;
Bmpr1acl/cl and littermate control skin revealed no
significant difference (average of 12±3 versus 12±2 follicles
per mm; P=0.766). At E16.5 and later embryonic stages, mutant
follicles were histologically similar to controls
(Fig. 4A,B and data not shown).
However, we found that the follicle density in E16.5 K14-Cre43;
Bmpr1acl/cl dorsal skin was significantly greater than in
control dorsal skin (7±2 versus 5±2 follicles per mm;
P=0.0002). As the sizes of mutant and control individuals were
similar at these stages, these results suggested that follicular morphogenesis
was accelerated in Bmpr1a mutants, but that the total number of
follicles that developed by birth was not affected by loss of epithelial
Bmpr1a.
|
Tooth development is arrested in epithelial Bmpr1a mutants
Surviving K14-Cre; Bmpr1acl/cl adult females
were able to nurse pups, indicating that functional mammary glands developed.
Histological analysis revealed that mammary buds and glands were present in
severely affected K14-Cre; Bmpr1acl/cl females at
E13.5 and birth, respectively, and whole-mount in situ hybridization with a
probe for the mammary bud marker Wnt10b revealed a normal pattern of
bud development at E13.5 (Fig.
4J-O). However, determining whether these mutants have subtle
defects in mammary morphogenesis will require further analysis.
By contrast, tooth development was strikingly affected in K14-Cre; Bmpr1acl/cl mice. Analysis of the histology of the oral cavity of K14-Cre43; Bmpr1acl/cl newborns revealed a complete absence of incisor or molar tooth structures (Fig. 4T,U). A dental lamina was present in K14-Cre43; Bmpr1acl/cl embryos at E13.5, and tooth bud formation was initiated. However, the tooth buds were significantly smaller and less well developed in mutant embryos than in controls (Fig. 4P,Q), and by E16.5 molar and incisor tooth structures had regressed (Fig. 4R,S and data not shown), indicating failure of tooth development after the bud stage.
Epithelial Bmpr1a is required for normal postnatal development of hair follicles
At later postnatal stages, dorsal and ventral skin of K14-Cre52;
Bmpr1acl/cl and K14-Cre40;
Bmpr1acl/cl mice, and mid-ventral skin of
bcre-32; Bmpr1acl/null mice, showed striking
defects in hair follicle morphology. By P8, control hair follicles were in
mid-anagen, with large hair bulbs that had descended deep into the fat layer
of the skin, and well-differentiated hair shafts that had undergone terminal
differentiation and lacked nuclei. By contrast, mutant mice displayed hair
follicles that were smaller, and were wavy and misangled. The mutant follicles
had not descended as deeply into the dermis as control follicles, and, perhaps
in part as a consequence of this, the fat layer appeared less expanded than
normal. Reduction of the fat layer probably also resulted from difficulties in
feeding caused by tooth defects in some mutants. The hair follicles had
misshapen, expanded dermal papillae, and the majority of follicles failed to
produce hair shafts. Instead, nuclei were present in the centers of the
abnormal follicles (Fig. 5A-D
and data not shown).
|
Epithelial Bmpr1a is required for hair shaft and IRS differentiation
To investigate whether the phenotype of mutant hair follicles resulted from
abnormal follicular differentiation we examined the expression of several
differentiation markers. Staining with AE13, an antibody to type I low-sulfur
hair-shaft cortex keratins (Lynch et al.,
1986), was absent in the majority of mutant follicles
(Fig. 5G,H). Staining with
AE15, which recognizes proteins in the medulla of the hair shaft and in the
IRS (O'Guin et al., 1992
), was
greatly reduced in the majority of follicles, and was absent from the most
distorted follicles, indicating absence of the hair-shaft medulla and
defective differentiation of the IRS (Fig.
5I,J). By contrast, the outer root sheath markers, keratins (K) 17
and 6, were expressed in mutant follicles, and weak K6 expression was observed
in mutant epidermis, suggesting epidermal hyperproliferation
(Ramirez et al., 1998
)
(Fig. 5K-N). These results
indicated that matrix cells failed to differentiate towards the hair shaft and
IRS in mutant follicles, and suggest a crucial role for BMP signaling in hair
follicle differentiation.
Transcriptional regulators of IRS and hair-shaft differentiation show reduced or absent expression in mutant follicles
Msx2 function is required for normal expression levels of
Foxn1, which controls the transcription of several hair keratin genes
(Ma et al., 2003;
Meier et al., 1999
;
Schlake and Boehm, 2001
).
Expression of Msx2, the related gene Msx1, and
Foxn1 was dramatically reduced in mutant skin
(Fig. 6A-D,I,J).
Differentiation of the IRS is regulated by the transcription factor GATA3
(Kaufman et al., 2003
), and
GATA3 expression was similarly reduced or absent in mutant follicles
(Fig. 6G,H). Expression of the
WNT pathway transcriptional effector Lef1 was maintained in mutant
follicles (Fig. 6K,L); however,
nuclear localization of ß-catenin, which complexes with LEF1 to activate
WNT target transcription, was absent from the epithelium of the most affected
follicles (Fig. 6M,N). Nuclear
LEF1 and ß-catenin localize to hair-shaft precursor cells and are thought
to regulate differentiation of the hair shaft
(DasGupta and Fuchs, 1999
;
Merrill et al., 2001
). Taken
together, therefore, these results indicate that BMPR1A is required for cells
exiting the matrix to begin to differentiate to form hair shaft and IRS.
Consistent with this role for BMPR1A, Shh, which regulates
proliferation in hair follicles (Chiang et
al., 1999
; Oro and Higgins,
2003
; St-Jacques et al.,
1998
), was expressed in mutant follicles
(Fig. 6E,F).
|
Mutant hair follicles fail to undergo programmed regression
To determine whether hair follicles cycle normally in the skin of surviving
mutant mice, we examined the histology of dorsal and ventral skin from
K14-Cre40; Bmpr1acl/cl mice at P12, P21 and P34,
and ventral skin from bcre-32; Bmpr1acl/null mice
at P17, compared with control littermates. At P12, control hair follicles were
in a fully developed anagen stage, with large hair bulbs and differentiated,
pigmented hair shafts (Fig.
7A). Mutant follicles had not descended further into the dermis
than they had at P8, and were still wavy and misangled
(Fig. 7B). At P17, control
follicles were entering catagen (Fig.
7C). By contrast, mutant hair follicles did not regress; instead
they persisted in an abnormal anagen phase
(Fig. 7D). At P21, control
follicles were in the telogen, resting phase, whereas mutant follicles were in
an abnormal, anagen-like state (Fig.
7E,F). The dermal papillae of mutant follicles, revealed by
staining for alkaline phopshatase, were expanded compared with those of
control littermate follicles (Fig.
7E',F'). Anti-BrdU immunofluorescence revealed high
levels of incorporation in the mutant hair follicle bulbs and outer root
sheaths at P21 (Fig.
7F''); by contrast, little proliferation was detected in
control littermate skin at this stage (Fig.
7E''). By P34, control follicles had re-entered anagen and
high levels of proliferation were detected in the follicle bulbs
(Fig. 7G,G'). At this
stage some less distorted mutant follicles were entering catagen. However, the
majority of mutant follicles had still not undergone regression, and cells in
the hair bulb and outer root sheath continued to proliferate
(Fig. 7H,H'). The mutant
epidermis also showed more proliferation than controls
(Fig. 7G',H').
TUNEL assays did not reveal differences in apoptosis between mutant and
control anagen stage follicles (not shown).
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Discussion |
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We observed accelerated embryonic hair follicle development in
Bmpr1a mutants, consistent with observations that loss of the BMP
inhibitor noggin inhibits hair follicle morphogenesis and with data indicating
that BMP signals suppress feather follicle fate
(Botchkarev et al., 2002;
Jung et al., 1998
;
Noramly and Morgan, 1998
). In
contrast to this relatively mild embryonic hair follicle phenotype, tooth
morphogenesis was highly abnormal in K14-Cre43;
Bmpr1acl/cl mice. A dental lamina was formed, and tooth
bud development was initiated; however, at E13.5 these structures were smaller
and less developed than in control littermate embryos, and were resorbed by
E16.5. These observations are consistent with a previously proposed role for
BMP signaling from the mesenchyme to the epithelium that is thought to
regulate transition from the bud to the cap stage of tooth development
(Jernvall and Thesleff,
2000
).
Our finding that deletion of Bmpr1a causes severe defects in
postnatal differentiation of the hair shaft and IRS is consistent with
upregulation of BMP pathway genes during anagen
(Botchkarev et al., 2001;
Kulessa et al., 2000
), and
with our observation that nuclear phopsho-SMAD accumulates in hair shaft and
IRS precursor cells. Similar defects in hair shaft and IRS differentiation
resulting from K14-Cre-mediated excision of Bmpr1a were
reported by Kobielak et al. while this manuscript was under revision
(Kobielak et al., 2003
), and
transgenic mice expressing the BMP inhibitor noggin under the control of an
Msx2 promoter also show defective hair-shaft differentiation
(Kulessa et al., 2000
). IRS
differentiation is relatively normal in Msx2-noggin mice, perhaps due
in part to the restricted domain of Msx2 promoter activity and to the
fact that noggin downregulates the Msx2 promoter and so is not
expressed at high levels.
In Bmpr1a mutant hair follicles, we observe absent or severely
decreased expression of the hair-shaft regulatory genes Foxn1, Msx1
and Msx2. This suggests that BMPR1A signaling may not activate
hair-shaft keratin gene expression directly, but instead by inducing
expression of Msx genes, which in turn activate Foxn1
(Ma et al., 2003). In
addition, we find that expression of GATA3, a transcription factor that
regulates differentiation of matrix cells into IRS
(Kaufman et al., 2003
),
requires epithelial BMPR1A, a result that was also obtained by Kobielak et al.
(Kobielak et al., 2003
).
Although hair-shaft keratins and several transcription factors associated
with terminal differentiation are strongly downregulated or not expressed in
Bmpr1a mutants, Lef1 expression was maintained in the matrix
of mutant follicles. Despite expression of Lef1, nuclear
translocation of ß-catenin, indicating activation of the WNT pathway, was
not observed in the epithelium of the most affected mutant follicles,
suggesting that WNT pathway activation lies downstream of epithelial BMPR1A
signaling. The recent identification of Foxn1 as a direct WNT target
gene (Balciunaite et al., 2002)
suggests that BMPR1A might regulate Foxn1 expression by dual
mechanisms: activation of WNT signaling and induction of Msx
expression.
In wild-type hair follicles, matrix cells cease proliferating as they rise up in the follicle beyond the level of the dermal papilla, and begin to express transcription factors that regulate differentiation of the hair shaft and IRS. In the absence of epithelial Bmpr1a, post-mitotic cells are observed in the upper follicle bulb (Fig. 7F'',H'; Fig. 8G), but these fail to express regulatory factors or to differentiate towards either hair shaft or IRS. Thus Bmpr1a is not required for exit from the highly proliferative matrix compartment, but regulates the next step: differentiation.
Interestingly, the more severely affected epithelial
Bmpr1a-deficient hair follicles do not undergo programmed regression,
but instead continue to proliferate, eventually forming follicular cysts and
matricomas. As nuclear localized phospho-SMAD is dramatically decreased at the
anagen-catagen transition, it is unlikely that epithelial BMPR1A plays a
direct role in initiating catagen onset. Instead, continued proliferation of
mutant follicles may indicate that, in the absence of terminal
differentiation, the factors that normally signal the end of anagen are not
produced. Alternatively, as we find that BMP signaling is active in the hair
follicle bulge during the resting phase, and ectopic noggin induces anagen
onset (Botchkarev et al.,
2001), it is possible that epithelial BMPR1A signaling normally
acts to suppress the proliferation of stem cells. Proliferation of these cells
in the absence of epithelial BMPR1A could potentially feed expansion of the
matrix, over-riding the signals that would normally induce catagen, and, in
the absence of follicular differentiation, leading to the development of
matricomas.
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ACKNOWLEDGMENTS |
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