1 Division of Genetics, Department of Medicine, Brigham and Women's Hospital and
Harvard Medical School, Boston, MA 02115, USA
2 Department of Cell and Molecular Biology, Tulane University, New Orleans, LA
70118, USA
3 Department of Pathology, University of Southern California, Los Angeles, CA
90033, USA
4 Center for Craniofacial Molecular Biology, University of Southern California,
Los Angeles, CA 90033, USA
5 Department of Dermatology, University Hospital Hamburg-Eppendorf, 20246
Hamburg, Germany
6 The Jackson Laboratory, Bar Harbor, ME 04069, USA
7 Department of Biochemistry, University of Southern California, Los Angeles, CA
90033, USA
* Authors for correspondence (e-mail: chuong{at}pathfinder.usc.edu and maas{at}rascal.med.harvard.edu)
Accepted 2 October 2002
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SUMMARY |
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Key words: Alopecia, Hair cycle, Hair differentiation, Homeobox genes, Msx2, Foxn1, Ha3, Fgf5, Mouse
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INTRODUCTION |
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Less is known about the cellular and molecular mechanisms that regulate the
differentiation and cycling of the hair follicle. During differentiation,
germinative matrix cells in the hair bulb (TA or transient amplifying cells)
actively divide to produce progenitor cells that differentiate and give rise
to the hair shaft and to the inner root sheath or IRS
(Rogers et al., 1998) (see
Fig. 1G). These cell types are
arranged in concentric layers from outside to inside, respectively. Several
pathways are involved in the specification and differentiation of matrix cells
into hair shaft and sheath cells. BMP signaling is required during hair
differentiation, as attenuation of BMP signaling by ectopic Noggin abolishes
hair filament differentiation but not that of the IRS
(Kulessa et al., 2000
). The
Wnt signaling pathway is also implicated in hair follicle morphogenesis, with
Lef1 constituting a key nuclear effector in this pathway
(Zhou et al., 1995
;
DasGupta and Fuchs, 1999
;
Millar et al., 1999
). Wnt
signals act on the dermal papilla and probably prompt the epidermis to induce
hair development (Kishimoto et al.,
2000
). Although Lef1 RNA is expressed in matrix cells
during anagen, Lef1 protein accumulates in the nucleus of postmitotic
precortex cells undergoing terminal differentiation
(DasGupta and Fuchs, 1999
).
Cell-cell interactions involving the Notch signaling pathway are also crucial
for proper hair differentiation (Powell et
al., 1998
; Lin et al.,
2000
). In addition, the homeobox gene Hoxc13 has been
implicated in the regulation of hair differentiation, as both overexpression
(Tkatchenko et al., 2001
) and
genetic ablation (Godwin and Capecchi,
1998
) lead to defective hairs and alopecia and HOXC13 can regulate
hair keratin promoters directly
(Jave-Suarez et al., 2002
).
Last, the nude gene product Foxn1 (also known as Whn) is implicated
in hair progenitor cell differentiation as matrix cells exit the cell cycle
and migrate up along the hair shaft en route to terminal differentiation
(Lee et al., 1999
). However,
the relationships between these different genes in hair differentiation are
unresolved.
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One of the more remarkable characteristics of the hair follicle biology is
its growth cycle, which includes anagen, catagen and telogen (reviewed by
Fuchs et al., 2001;
Stenn and Paus, 2001
) (see
Fig. 1G). Anagen is the stage
of active cell proliferation and differentiation. During anagen, the follicle
lengthens and penetrates into the dermis. Meanwhile, descendants of the hair
matrix cells at the base of the follicle bulb are gradually pushed upwards,
differentiating into the hair shaft that emerges from the follicle. In
catagen, hair production ceases and the hair bulb region is converted into an
anchoring club with a thin epithelial strand connecting the inner root sheath
(Hardy, 1992
;
Koch et al., 1998
). Cellular
apoptosis in follicular keratinocytes is associated with catagen progression
(Lindner et al., 1997
). By the
end of catagen, the dermal papilla relocates to the vicinity of the hair
follicle bulge. Telogen is mainly the resting stage, but an exogen stage also
has been defined that represents the stage of controlled hair shaft-extrusion
(Paus et al., 1999
). After
telogen, the next round of hair growth begins with interaction between the
dermal papilla and the multipotent epithelial stem cells that reside in the
outer root sheath (ORS) and in the bulge region
(Cotsarelis et al., 1990
;
Oshima et al., 2001
). Through
these interactions, a group of TA cells are induced and anagen restarts when
new follicle pushes old one to the side. At some point in telogen the old
fiber (club hair) is lost. Several molecules and diffusible growth factors
exhibit oscillatory expression during different phases of the hair cycle, and
transgenic overexpression of some of these leads to acceleration or arrest of
the hair cycle (reviewed by Paus et al.,
1999
; Stenn and Paus,
2001
). However, only a few genes have been shown to directly
influence hair cycling.
One family of genes involved in signal transduction between interacting
tissue layers during organogenesis is the mammalian Msx homeobox
family, homologs of the Drosophila msh (muscle segment homeobox) gene
(for a review, see Davidson,
1995). During mouse embryogenesis, Msx1 and Msx2
are expressed in hair follicle placode ectoderm, and subsequently in
epithelial matrix cells. Both genes are expressed during anagen in the matrix
cells of the hair bulb (Reginelli et al.,
1995
). Mice doubly homozygous for Msx1 and Msx2
have
1/3 the wild-type number of induced follicles, and these incipient
follicles are associated with reduced Patched expression, suggesting
a threshold requirement for Msx protein in hair follicle induction
(Satokata et al., 2000
).
Whereas transgenic mice overexpressing Msx2 under the cytomeglovirus
(CMV) promoter exhibit retarded hair growth and a reduced hair matrix
(Jiang et al., 1999a
),
preliminary analysis of Msx2-deficient hair follicles suggests that
catagen onset occurs prematurely (Satokata
et al., 2000
). However, the mechanisms by which Msx2 acts
during the mouse hair growth cycle and by which its deficiency leads to hair
loss are unclear. Msx2 knockout mice exhibit a curious balding
phenotype, characterized by repeated cycles of hair loss and regrowth in
different domains of the body, which we named cyclic alopecia. We analyzed its
pathogenesis and revealed defects in all hair cycle stages hair
differentiation.
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MATERIALS AND METHODS |
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In situ hybridization
Radioactive in situ hybridization was performed by hybridizing
-[35S]-labeled cRNA probes to skin sections as described
(Wawersik and Epstein, 2000
;
Maas et al., 1996
;
Zhang et al., 1999
).
RNase protection assays
Whole-cell RNA was prepared from dorsal skin of wild type and Msx2
knockout mice at different stages during the first anagen using the Ultraspec
RNA isolation system (Biotecx Laboratory, TX). Antisense probes for
Bmp4 and Lef1 were generated as described
(Chen et al., 1996). A
Tgfa probe was generated by PCR using primers Tgfa-F
(5'-TGTCAGGCTCTGGAGAACAGC-3') and Tgfa-R
(5'-CGGCACCACTCACAGTGTTTG-3') on reverse transcribed skin total
RNA. The resulting 350 bp fragment was cloned into the SmaI site of
pGEM4Z (Promega, WI). An antisense probe for Tgfa was generated by
digesting the above plasmid with EcoRI followed by in vitro
transcription with T7 RNA polymerase. The Foxn1 antisense RNA probe
was generated by linearizing pBSK-Foxn1 with XhoI followed
by transcription with T3 polymerase (Nehls
et al., 1994
). RNA probes for ß-actin and RPL19 were
respectively prepared according to manufacturer's instructions (Ambion, TX) or
as described (Ma et al.,
1998
). Total skin RNA (20 µg) was hybridized overnight at
45°C with 4x105 cpm of
- [32P]-UTP
labeled antisense RNA probes for Bmp4, Lef1, Ha3, Tgfa or
Foxn1, with ß-actin or Rpl19 as loading controls. After
digestion with 20 µg/ml RNase A and 1.5 µg/ml RNase T1, protected
fragments were precipitated and separated on a 6% denaturing polyacrylamide
gel and band intensities were quantified by phosphorimager (Molecular
Dynamics).
Cell proliferation and apoptosis assay
Mice were injected subcutaneously with 50 µg/g body weight of BrdU at a
concentration of 5 mg/ml in PBS. Anti-BrdU-peroxidase antibody staining was
processed according to the manufacturer's instructions (Boehringer Mannheim,
#1585860). TUNEL staining of dorsal skin of P45 mice was performed using the
In Situ Cell Death Detection Kit (Boehringer Mannheim, #1684809).
Immunohistochemistry
Primary antibodies used were rabbit/mouse anti-Foxn1
(Lee et al., 1999), rabbit
anti-K14 (1:400, Berkeley Antibody Company); anti-K10 (1:200, Sigma),
monoclonal AE 13, 14, 15 (gift of Dr T. T. Sun)
(Lynch et al., 1986
).
Immunohistochemistry was performed using the peroxidase substrate kit (Vector
Laboratories, Inc.) or immunofluorescence as described in the manufacturer's
instructions.
Hair stripping
Hair stripping experiments were performed by coating the dorsal skin of P45
mice with wax at 60°C. Wax was pulled off along with hairs from the dorsal
skin after the wax had cooled to room temperature.
Scanning electron microscopy (SEM)
Hairs from the dorsal skin of mice were attached to carbon adhesive tabs on
aluminum mounts and coated with a Polaron sputter coater. Samples were
analyzed by scanning electron microscope at 20 kV
(Bechtold, 2000).
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RESULTS |
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Cyclic alopecia in Msx2 knockout mice
Msx2-deficient mice were analyzed on both the segregating C57BL/6J
and congenic ICR Backgrounds, with only slight differences in phenotype.
Newborn Msx2 knockout mice could be distinguished from wild-type
littermates by their short, curly vibrissae at P5. At P3, homozygous mutants
began to exhibit retarded pelage hair growth. Although control hairs were
straight, mutant hairs were wavy and exhibited irregular diameters along the
hair shaft (Fig. 2; see
Fig. 5). Measurements showed
that mutant hairs were 25-30% shorter, which was not related to the slight
overall growth retardation of these mutant mice. All four hair types (awl,
auchene, guard and zigzag) are present in Msx2 knockout mice.
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In contrast to heterozygous and wild-type littermates, at P14,
Msx2 knockout mice began to lose hair. This is followed by
progressive hair loss and regrowth. The mouse showed dynamic patches of
hairiness and baldness. The distinct appearance is illustrated by the two
pictures of the same mouse, taken one month apart
(Fig. 2A). To analyze this
phenomenon carefully, we observed the pattern of alopecia of five homozygote
mice twice a week over 3 months (Fig.
2B). The hair cycle-dependent hair loss helped to bring out the
concealed mouse skin domains that cycle asynchronously. Although hairs in
different domains cycle independently, hairs within the same domain cycle in a
synchronized wave, generally from anterior to posterior, but there are
occasions when this direction is reversed
(Fig. 2B). Similar asynchronous
hair cycling domains have been shown in normal mice
(Militzer, 2001).
In addition, in Msx2 knockout mice maintained on a C57BL/6J
segregating background, synthesis of the skin pigment melanin during the hair
cycle was also deranged (Fig.
2). Neural crest-derived melanocytes proliferate and mature during
anagen, subsequently undergoing apoptosis during early catagen (reviewed by
Tobin and Paus, 2001). In
Msx2 knockout mice, melanin synthesis commenced in early anagen, but
pigment was lost in regions of hair loss, suggesting that hair follicles in
bald regions prematurely enter catagen.
Abnormal hair cycling in Msx2 knockout mice
To analyze hair cycling in Msx2-deficient hair follicles, we
examined mid-dorsal skin histology at the level of thoracolumbar junction
between wild-type littermates and Msx2tm1Rilm homozygous
mice at 11 serial time-points between P1 and P31
(Fig. 3, left column). Although
no histological differences were observed at P1, by P3 Msx2-deficient
skin had a thinner dermis. At P5, wild-type follicles were in anagen VI when
all follicular cell types are present and the follicles penetrate into the
adipose layer. By contrast, Msx2-deficient skin had a thinner dermis
and a significant region of the hair follicles were horizontal, parallel to
the muscle layer (Fig. 3B,
arrowheads). This may reflect asynchrony between the down growth of hair
follicles and the ability of the dermis to accommodate them. At P10, when
wild-type mice still possessed anagen hair, hair in Msx2 knockout
mice had already entered catagen. However, by P17, when wild-type mouse skin
displayed catagen V-VII follicles, Msx2-deficient skin displayed
early abnormal catagen II follicles characterized by small and long hair bulbs
(data not shown). Moreover, by P21, wild-type mice displayed only telogen
follicles, whereas Msx2 knockout mice displayed abnormal catagen
VI-VII follicles, further indicating a significant delay in catagen
progression.
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To confirm this result, we examined the expression of Tgfa, a
marker for the inner root sheath (IRS) and keratogenous zone
(Luetteke et al., 1993). By
P17, the number of Tgfa-expressing follicles was much reduced, and no
Tgfa expression could be detected in wild-type P21 telogen hair
follicles. By contrast, in Msx2-deficient hair follicles
Tgfa continues to be strongly expressed at P21, consistent with a
prolonged catagen (Fig. 3E,F,
inset). At P24, control skin has re-entered anagen; however, hairs in
Msx2-deficient skin remain in telogen until about P31, when they are
seen to re-enter anagen.
We also quantify the results by histograms that show the percentage of hair follicles in anagen, catagen, and telogen phases in different ages of the mice. These results suggest that in Msx2-deficient skin, there is a shortened anagen phase, followed by an abnormal and prolonged catagen phase. The shortened hair in Msx2 knockout mice is also consistent with the conclusion that anagen is shortened.
To challenge the ability of Msx2-deficient hair follicles to re-enter anagen, we carried out wax stripping of hairs to synchronize hair cycling (Fig. 3, right column). Six days after stripping, anagen hair follicles first re-appeared in wild-type skin, and by 20 days after stripping, these hair follicles finished their cycling and entered telogen. By contrast, in Msx2-deficient skin, anagen follicles did not re-appear until 20 days after stripping, suggesting that hairs in Msx2 knockout mice have difficulty re-entering anagen phase. Measurement of percentages of hair follicles stages showed this clearly. Taken together, we conclude that while Msx2-deficient hair follicles can complete the hair cycle, their cycles are out of phase compared with normal hair follicles because of abnormalities in transitions between all three major phases of the hair cycle.
Msx2 and Fgf5 in the genetic pathway controlling
the hair cycle
As Msx2 knockout mice have a shortened anagen, we used a well
characterized spontaneous mutant mouse mutant, the angora
(Fgf5go/Fgf5go) mouse, which is
Fgf5-deficient and has an abnormal prolongation of anagen VI
(Hébert et al., 1994;
Sundberg et al., 1997
). To
probe the interactions between these genes, mice deficient for both
Fgf5 and Msx2 were generated by crossing the mutant stocks.
Compound heterozygotes were phenotypically indistinguishable from wild type
and were crossed to generate Fgf5go, Msx2tm1
Rilm double homozygotes. Examination of 48 Msx2 knockout
mice revealed that hair loss always occurred at P14, regardless of genetic
background (Fig. 4A,B).
Msx2tm1 Rilm homozygotes carrying one
Fgf5go allele exhibited the same cyclic alopecia phenotype
as Msx2tm1 Rilm homozygous only. However, mice doubly
homozygous for Msx2 and Fgf5 no longer lost their pelage
hair at P14 (Fig. 4A,B).
Despite the initial delay in hair growth in Msx2tm1 Rilm
homozygous mice, Fgf5go, Msx2tm1 Rilm double
homozygotes grew long pelage hairs characteristic of the angora
(Fgf5go) mutation by P16. Between P18 and P30, all double
homozygotes eventually lost their pelage hairs
(Fig. 4B,C). The variation in
the timing of hair loss in Msx2, Fgf5 double knockout mice could
reflect variability in genetic background, as the hair cycle phases of
different strains of mice have different lengths
(Hébert et al., 1994
).
In angora mice, it has been shown that anagen is extended by 3 days
(Pennycuik and Raphael, 1984
;
Sundberg et al., 1997
). In
Msx2 knockouts, hair loss is temporally linked to a specific point of
time in catagen; the observation that both catagen onset and hair loss are
postponed by 4 or more days in Fgf5go, Msx2tm1
Rilm double mutants (for Msx2 mutants, the C57BL/6J strain
was used) further supports the idea that hair loss in Msx2 knockout
mice is associated with catagen onset. It also suggests that the consequence
of defective Fgf5 is dominant compared with those of mice with
defective Msx2. However, in Msx2 knockout mice,
Fgf5 in the ORS (Hébert et
al., 1994
; Rosenquist and
Martin, 1996
) was not significantly altered in anagen (data not
shown). So the two pathways may not interact directly.
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Defective hair shaft differentiation in Msx2 knockout
mice
If a hair cycle defect was the only abnormality in Msx2 knockout
mice, the hair length would vary but the hair should not be lost. Hair loss
can be the result of breakage at the shaft region because of structural
defect, or the loss of the club hair due to defect of the follicle to retain
the club hair. To investigate the mechanism of hair loss further, we examined
the structure of the hair shaft medulla, cortex and cuticle
(Fig. 5A-J). Msx2
knockout mice hairs were short, wavy and of uneven diameter, and their
medullae contained disorganized septates, septulates and air bubbles
(Fig. 5A-D,I,J). SEM analyses
showed that, unlike the scaly tile pattern of the cuticle covering the surface
of wild-type hairs, Msx2 knockout mice hairs exhibited flattened
cuticles with wrinkled surfaces (Fig.
5E-H) (Rogers et al.,
1998). To differentiate whether hair loss is due to breakage of
the shaft or dislodge of the club hair, we examined the plucked hairs. We
observed that these hairs have their club ends, and none breaks in the middle.
Application of the tape stripping test
(Koch et al., 1998
) showed
that mutant hairs were much more easily removed than wild-type hairs. Although
the club end appears to be morphologically similar between normal and mutant
hairs (Fig. 5I,J), defects in
the cuticle or the formation of the club are likely to contribute to the loose
hair attachment between the club hair and the inner root sheath, resulting in
the hair loss observed during catagen.
The short abnormal hair shaft could result from either defective proliferation or differentiation of the hair matrix cells. We first examined whether matrix cell proliferation was affected in Msx2 knockout mice anagen hair follicles by injecting BrdU into P5, P9, P11 and P15 Msx2 knockout mice and wild-type littermates, collecting skin samples 2 hours after injection. At P11, germinative matrix cells in both wild-type and Msx2 knockout mice anagen hair follicles showed similarly extensive cellular proliferation in the lower region of the hair bulb and outer root sheath (Fig. 5K,L). We quantified cellular proliferation by counting BrdU-positive cells in serial sections through the germinative matrix of 12 histologically similar follicles in Msx2 knockout mice and wild-type littermates. The average number of BrdU-positive cells per follicle section (±s.e.m.) was not significantly different between Msx2 knockout mice [34.6±7.6 (BrdU+ cells/follicle section)] and wild-type littermates [32.5 ± 6.3 (BrdU+ cells/follicle section)]. Similarly, no significant differences in matrix cell proliferation were observed between Msx2 knockout mice and wild-type littermates at P5, P9 and P15 (data not shown).
TUNEL-positive cells were found in hair follicles. They are associated with
catagen and also other hair morphogenesis processes
(Lindner et al., 1997).
Examination of cellular apoptosis was also carried out on P5, 9, 11, 14 and 30
wild-type and Msx2 knockout mice skin sections. No apparent
difference was detected and an example is shown
(Fig. 5M,N).
To assay differentiation, we used immunohistochemistry and in situ
hybridization to detect molecules that represent different layers of hair
related epidermis. Keratin 10 (K10) is expressed in the suprabasal layer of
the interfollicular epidermis and K14 in the basal layer and outer root
sheath. There were no detectable changes relative to wild type (data not
shown). Immunohistochemistry with AE13 (react with low sulfur hair keratin)
and AE14 (react with high sulfur hair keratin) monoclonal antibodies were
expressed in the hair cortex (Lynch et
al., 1986), and reduced in the mutants (not shown). Levels of
mouse acidic (type I) hair keratin (Ha3; Krt1-2 Mouse Genome
Informatics), another marker for hair cortex cells
(Winter et al., 1994
;
Meier et al., 1999
), were also
reduced (Fig. 6A,B). A similar
increase in the expression of Ha3 transcripts from P5 to P11 followed
by a decrease was observed in both wild-type and in Msx2-deficient
skin, but in mutants the levels were reduced to 31% from 72%
(Fig. 6A,B). These results
indicate a failure of shaft differentiation in Msx2-deficient hair
follicles.
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The Foxn1 regulatory pathway is altered in Msx2
deficient hair follicles
We next focused on signaling pathways that might be affected by
Msx2 deficiency. Ha3 was shown to be a downstream target of
Foxn1 (Meier et al.,
1999; Schorpp et al.,
2000
). As Ha3 is much reduced in Msx2 knockout
mice, we examined whether Foxn1 also resides downstream of
Msx2. Like Msx2, Foxn1 is highly expressed in the hair
cortex with lower levels in matrix and outer root sheath cells
(Lee et al., 1999
;
Meier et al., 1999
).
Foxn1 expression was analyzed serially by RNase protection between P1
and P17 in wild-type and Msx2-deficient skin and was expressed at all
days after P1. However, in Msx2-deficient skin, Foxn1
expression was markedly decreased at all time points
(Fig. 6A,B). Quantification of
6 sets of RNase protection assays at P11 revealed a 50% reduction in
Foxn1 expression in Msx2 deficient skin
(Fig. 6B). Furthermore,
immunostaining with an affinity purified polyclonal anti-Foxn1 antibody
detected Foxn1 protein in the hair matrix and pre-cortex, with a relative
enrichment in the cortex regions in wild-type follicles. This immunoreactivity
was significantly reduced in Msx2 knockout mice
(Fig. 6C), consistent with the
conclusion that Foxn1 expression requires Msx2 function. To see
whether Foxn1 is downstream to Fgf5, we examined the
expression of Foxn1 in Fgf5-deficient mice. We did not
observe difference in expression level or distribution pattern (not shown). We
also examined the expression level of Lef1 and Bmp4. While
no significant reduction of Bmp4 is detected, we observed a
consistent low level reduction of Lef1 mRNA.
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DISCUSSION |
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The role of Msx2 in regulating the hair cycle
Recently, both signaling molecules and transcription factors that exhibit
altered expression during different cycle phases have been identified.
Classical experiments suggest the existence of an intrinsic control of the
hair cycle that resides in the hair follicle itself, and that can be modulated
by outside factors (reviewed by Stenn and
Paus, 2001). In mouse hair follicles, it has been proposed that
such an intrinsic biological clock, termed the `hair cycle clock', controls
the length of each phase of the hair cycle
(Paus et al., 1999
;
Stenn and Paus, 2001
).
Although the molecular nature of this clock is unknown, it may involve
hormones or diffusible factors whose expressions oscillate in each hair cycle.
This view implies that the hair cycle is controlled by sets of molecules that
coordinately regulate the anagen-catagen, catagen-telogen and telogen-anagen
transitions. According to this model (Fig.
7) sets of regulatory factors act at each checkpoint either to
promote or suppress the transitions between the phases of the hair growth
cycle. The observed length of each phase thus reflects the balanced strength
of the promoting and suppressing factors impinging on each transition point.
Analysis of the hair loss phenotype in Msx2 mutant mice permits the
integration of Msx2 into this model.
|
In wild-type hair follicles, catagen is accompanied by changes in follicle
morphology, including cessation in matrix cell proliferation and apoptosis in
the hair bulb. However, in Msx2-deficient hair follicles, these
events are uncoupled. As early as P10, Msx2-deficient hair follicles
exhibit histological signs of catagen, while those of heterozygous littermates
remain in anagen. However, matrix cells in both wild-type and
Msx2-deficient follicles show comparable numbers of BrdU-positive
cells. Therefore, there is an apparent uncoupling between proliferation and
differentiation in the Msx2 deficient hair follicle. The precocious
onset of catagen in Msx2 knockout mice suggests that Msx2
normally plays a role in maintaining hair follicles in anagen. Such a role is
consistent with the role proposed for Msx genes in maintaining the
proliferative potential of various cell populations (e.g. hair matrix cells)
and in preventing their differentiation
(Hu et al., 2001). Pursuant to
this view, Msx2 would be predicted to suppress the anagen-catagen
transition. In the model presented in Fig.
7, loss of Msx2 could thus either weaken the suppressive
influences or enhance the positive influences converging at anagen-catagen
transition, thereby facilitating catagen entry. At the same time, the
prolongation of catagen suggests that Msx2 also plays a positive role
in the transition to telogen. Similarly, Msx2 is also required for
anagen re-entry, as Msx2 knockout mice exhibit a 10-day delay in
re-entering anagen after hair stripping. Premature hair loss in Msx2
knockout mice is not due to hair shaft breakage, but the result of the loss of
whole club hairs. Therefore, there is a precocious entry of exogen.
Several growth factors have been implicated in the control of catagen
onset, and we took advantage of one of these,
Fgf5go/Fgf5go, to dissect further the genetic
pathway controlling catagen entry
(Hébert et al., 1994).
Fgf5 is expressed in the ORS during late anagen and has been
suggested to induce catagen by diffusion into the dermal papilla
(Rosenquist and Martin, 1996
).
To test their relationship, we crossed Fgf5 knockout mice, which have
long hairs as a result of a prolongation of anagen VI, into the
Msx2-deficient background to generate mice doubly homozygous for
Fgf5go and Msx2tm1Rilm. These mice
also had long hairs, and while these hairs were eventually lost, the timing of
hair loss was significantly delayed, suggesting an elongated anagen phase
consistent with the angora phenotype. However, expression of Fgf5 is unaltered
in Msx2 knockout mice. These results suggest that Fgf5 and
Msx2 may act independently in their regulation of the length of
anagen.
Msx2 is required for hair shaft differentiation
Both development of the hair follicle and subsequent hair growth involve
signaling between the dermal papilla and matrix cells. During anagen, TA cells
in the hair matrix proliferate in response to growth signals from the dermal
papilla, differentiating into the several hair cell types of the hair shaft
and IRS (Fig. 7). The fate of
these epithelial cells may be determined as soon as they leave the cell cycle,
or later during differentiation via cell-cell interactions
(Kopan and Weintraub, 1993).
Regulation of this process has been proposed to involve FGFs, BMPs
(Kulessa et al., 2000
), Notch
(Lin et al., 2000
), Foxn1
(Prowse et al., 1999
) and Wnt
(Millar et al., 1999
)
(Fig. 7). As Msx2
deficiency causes defects in all three layers of the hair shaft, we consider
the role of Msx2 is to integrate the differentiation of TA cells, as
Msx2 is expressed in regions where proliferating matrix cells migrate
up to become precortical cells and subsequently differentiate into hair cortex
cells.
Differentiation of hair cortex cells requires functional Foxn1 protein, a
winged helix transcription factor. Loss of Foxn1 function gives rise
to the nude mouse phenotype in which differentiation of hair progenitor cells
is severely affected, resulting in short, bent hairs that rarely protrude
beyond the skin surface where they break off
(Nehls et al., 1994;
Brissette et al., 1996
).
Recently, Foxn1 has been shown to be a transcriptional regulator of the mouse
acidic hair keratin genes, providing one of the molecular mechanisms that may
contribute to the nude phenotype (Meier et
al., 1999
; Schorpp et al.,
2000
; Baxter and Brissette,
2002
). The acidic hair keratin Ha3 is specifically expressed in
the hair cortex layer and thus serves as a terminal differentiation marker.
Interestingly, downregulation of Ha3 and decreased immunoreactivity
of low and high sulfur hair keratins are observed in Msx2-deficient
hair, consistent with the observed structural defect in cortex cell
differentiation. These results suggest that an Msx2/Foxn1/Ha3 pathway
participates in the control of hair shaft growth and differentiation
(Fig. 7). However, the lost
hairs show a complete length, suggesting that defect is due to weakness in
retaining club hairs, not due to breakage of the shaft.
What factors reside upstream of Msx2 in the matrix and precortex
region? During hair differentiation, Bmp4 is expressed in hair matrix
cells and in hair shaft cells in contact with the IRS. Bmp2 is
specifically expressed in the precortex cells
(Lyons et al., 1991;
Kulessa et al., 2000
), while
noggin is expressed in the dermal papilla
(Botchkarev et al., 1999
).
Ectopic expression of noggin in the hair matrix under a minimal Msx2
promoter disrupts hair differentiation, with the cells remaining in a highly
proliferative state in the precortex and hair shaft regions; these results
provides strong evidence that BMPs are required during hair differentiation
(Kulessa et al., 2000
).
Msx2 expression is markedly reduced in these Msx2-noggin
mice. Conversely, we find that Bmp4 expression is preserved in
Msx2 mutant skin. In addition, as the defects in
Msx2-deficient hair follicles are mainly restricted to the hair shaft
and are less severe than those associated with abolition of Bmp signaling
(which involves both IRS and hair shaft), Msx2 is likely to function
downstream of Bmp genes during hair differentiation.
Another major pathway involved in hair morphogenesis is the Wnt/catenin
pathway. ß-Catenin participates in the maintenance of epidermal stem
cells, and activation of ß-catenin can induce new skin appendages in
mouse and chicken (Chan et al.,
1999; Gat et al.,
1998
; Widelitz et al.,
2000
; Kishimoto et al.,
2000
; Huelsken et al.,
2001
). Msx2 is co-expressed in hair matrix cells with
Lef1, a key effector in the Wnt/catenin pathway. High levels of
Lef1 expression are found in proliferating matrix cells, possibly
transducing Wnt signals. Later, nuclear Lef1 protein accumulates in pre-cortex
cells undergoing terminal differentiation
(DasGupta and Fuchs, 1999
),
and several keratin promoters contain Lef1-binding sites
(Zhou et al., 1995
). These
results suggest that Lef1 is required to prepare matrix cells for terminal
differentiation. The reductions in Lef1 expression we observe in
Msx2-deficient hair follicles are modest, but may indicate that
Lef1 resides downstream of Msx2 in matrix and pre-cortical
cell differentiation, similar to the genetic relationship proposed between
Msx1 and Lef1 in tooth development
(Chen et al., 1996
;
Kratochwil et al., 1996
).
An intricate signaling network in the hair matrix region regulates the specification and differentiation of the hair shaft and the IRS. From the analyses performed to date, Msx2 is unlikely to be the more upstream molecules in this network, but it is likely to constitute a key regulator in the differentiation of TA cells into hair shaft cells. For example, upon Msx2 deficiency, pre-cortical cells may fail to respond fully to signals such as BMPs, Wnts, Fgfs or Notch ligands. As a result, Foxn1 and Lef1 may fail to be expressed at the levels needed to ensure proper cortex differentiation and expression of hair keratins. The successful formation of a hair depends on the progression of hair progenitor cells through several major determination and morphogenetic events. Our results show that Msx2 is involved in regulating the switches between TA cells and pre-cortical cell and in the transitions between the different phases of the hair cycle.
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ACKNOWLEDGMENTS |
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