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First published online March 30, 2004
doi: 10.1242/10.1242/dev.01052
Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
Author for correspondence (e-mail:
fiona.watt{at}cancer.org.uk)
Accepted 22 December 2003
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Nß-cateninER).
Nß-cateninER was expressed in the epidermis of transgenic mice
under the control of the keratin 14 promoter and ß-catenin activity was
induced in adult epidermis by topical application of 4-hydroxytamoxifen
(4OHT). Within 7 days of daily 4OHT treatment resting hair follicles were
recruited into the hair growth cycle and epithelial outgrowths formed from
existing hair follicles and from interfollicular epidermis. The outgrowths
expressed Sonic hedgehog, Patched and markers of hair follicle
differentiation, indicative of de novo follicle formation. The interfollicular
epidermal differentiation program was largely unaffected but after an initial
wave of sebaceous gland duplication sebocyte differentiation was inhibited. A
single application of 4OHT was as effective as repeated doses in inducing new
follicles and growth of existing follicles. Treatment of epidermis with 4OHT
for 21 days resulted in conversion of hair follicles to benign tumours
resembling trichofolliculomas. The tumours were dependent on continuous
activation of ß-catenin and by 28 days after removal of the drug they had
largely regressed. We conclude that interfollicular epidermis and sebaceous
glands retain the ability to be reprogrammed in adult life and that continuous
ß-catenin signalling is required to maintain hair follicle tumours.
Key words: Stem cells, Differentiation, Trichofolliculoma, ß-catenin
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). It
is not only a complex, but also a very dynamic, tissue because there is rapid
cell turnover in the interfollicular epidermis (IFE) and hair follicles
undergo cyclic phases of growth (anagen), regression (catagen) and rest
(telogen). Maintenance of the epidermis depends on a population of
multipotential stem cells that have the ability to generate all of the
differentiated epidermal lineages (Niemann
and Watt, 2002). The epidermis is a frequent target for oncogenic
mutations and it is thought that stem cells are responsible for the production
of most epidermal tumours because, as long-term tissue residents, they can
accumulate multiple mutations and undergo clonal expansion
(Perez-Losada and Balmain,
2003; Owens and Watt,
2003).
In recent years there has been tremendous progress in identifying the
genetic and molecular changes occurring during malignant transformation.
Abnormal ß-catenin signalling, resulting from genetic alterations that
act by stabilising ß-catenin, have been implicated in many cancers,
including colorectal and hepatocellular cancer, melanoma and tumours of hair
follicles (reviewed by Polakis,
2000). ß-catenin is a structural component of adherens
junctions, linking cadherins to the actin cytoskeleton, and is also the key
effector of Wnt signalling, which plays a role during development of many
tissues (Peifer and Polakis,
2000). In the absence of Wnt, the cytoplasmic pool of
ß-catenin that is not complexed with cadherins is rapidly phosphorylated
at the N terminus by glycogen synthase kinase 3ß (GSK-3ß) and
ubiquitinated, resulting in its degradation. Wnt, through its receptor
frizzled, inhibits GSK-3ß, so that ß-catenin accumulates in the
cytoplasm and eventually translocates to the nucleus
(Henderson and Fagotto, 2002).
There it interacts with the N terminus of transcription factors of the Tcf/Lef
family and regulates transcription of target genes
(Huelsken and Birchmeier,
2001; Brantjes et al.,
2002; Moon et al.,
2002).
Several Wnts and their Frizzled receptors are expressed in the epidermis in
a highly dynamic and complex pattern
(Reddy et al., 2001). In
postnatal epidermis two members of the Tcf/Lef family are expressed: Tcf3 in
the bulge and outer root sheath (ORS) of the hair follicle, and Lef1 in the
ORS and matrix cells (DasGupta and Fuchs,
1999; Merrill et al.,
2001). Using an artificial promoter constructed of multimeric
Lef1/Tcf binding sites as a reporter of Wnt responsive cells, promoter
activity is observed during hair follicle formation in embryonic skin and
postnatally, both in the hair follicle bulge, a reservoir of stem cells, and
in hair shaft precursor cells (DasGupta
and Fuchs, 1999).
There is considerable evidence that ß-catenin signalling is important
in epidermal development, homeostasis and disease (reviewed by
Fuchs et al., 2001). Mice
expressing stabilised, N-terminally truncated ß-catenin under the control
of the keratin 14 (K14) promoter produce an excess of hair follicles and
develop tumours similar to human pilomatricomas and trichofolliculomas
(Gat et al., 1998). In humans
these benign tumours are associated with activating mutations of
ß-catenin (Chan et al.,
1999). Conversely, if ß-catenin is conditionally deleted in
the skin during embryogenesis there is no hair formation, and if the deletion
occurs after birth the hair is lost after the first hair cycle
(Huelsken et al., 2001). Hair
follicle development is also prevented when Dickkopf 1, an inhibitor of Wnt
action, is ectopically expressed (Andl et
al., 2002) or when the Lef1 gene is ablated in the epidermis
(van Genderen et al., 1994).
Transgenic mice in which the Wnt pathway is blocked by expressing N-terminally
truncated Lef1 in the epidermis have progressive hair loss and develop
epidermal cysts with interfollicular and sebocyte differentiation
(Merrill et al., 2001;
Niemann et al., 2002). When a
stabilised form of ß-catenin lacking the C-terminal transactivation
domain is expressed in the epidermis hair differentiation is promoted in the
interfollicular epidermis and hair follicles develop cysts of interfollicular
epidermis; this appears to reflect the status of the endogenous
ß-catenin/Tcf/Lef complexes in the cells
(DasGupta et al., 2002).
These observations clearly establish that ß-catenin plays a
fundamental role in morphogenesis of the hair follicle and strongly suggest
that the level of ß-catenin signalling determines whether keratinocytes
differentiate along the hair or interfollicular/sebocyte lineages. However,
the keratin 14 and keratin 5 promoters used for many of the transgenic mouse
studies are active during embryogenesis and indeed K5 and K14 expression is
detected at E9.5, as early as the single layered ectodermal stage
(Byrne et al., 1994). This
raises the possibility that the epidermis is only competent to respond to
altered ß-catenin signalling in postnatal life if it has undergone some
form of reprogramming during embryogenesis, analogous to T cell priming
(Melief, 2003). Indeed the
major effect of transient activation of ß-catenin in adult epidermis
appears to be to promote anagen (growth) phase of the hair cycle
(Van Mater et al., 2003). In
addition, experiments with cultured human interfollicular epidermis suggest
that ß-catenin may act primarily to alter stem cell number rather than to
direct lineage commitment: the putative stem cells express high levels of
cytoplasmic ß-catenin, and expression of stabilised ß-catenin
expands the stem cell compartment, while inhibition of ß-catenin
signalling promotes exit from the stem cell compartment
(Zhu and Watt, 1996;
Zhu and Watt, 1999). The
concept that ß-catenin might act primarily to control stem cell number
has recently received support from studies on haemopoiesis
(Reya et al., 2003).
The goal of our experiments was to establish whether activation of
ß-catenin signalling exclusively in adult epidermis is sufficient to
induce de novo hair follicle formation and hair follicle tumours. In addition
we wanted to obtain information as to the timing and duration of
ß-catenin signals required for these effects. We used the K14 promoter to
drive expression of a chimeric protein in which N-terminally truncated
ß-catenin is fused with an engineered form of the ligand binding domain
of the oestrogen receptor (ER), which is insensitive to endogenous oestrogen
(Littlewood et al., 1995). In
this way, the site and timing of ß-catenin activation is easily
controlled by topical application of the synthetic steroid 4-hydroxytamoxifen
(4OHT) to the skin of transgenic mice
(Arnold and Watt, 2001;
Van Mater et al., 2003).
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Nß-cateninER construct was generated by in frame insertion
of N-terminally truncated ß-catenin cDNA (nucleotides 715-2604, T2)
(Funayama et al., 1995;
Zhu and Watt, 1999) into the
BamHI site of the retroviral vector pBabepuro
(Morgenstern and Land, 1990)
containing the hormone-binding domain of a mutant murine oestrogen receptor
(Littlewood et al., 1995).
To generate the transgene construct, Nß-cateninER was excised
from pBabepuro, blunt-ended with Klenow DNA polymerase and cloned into the
blunt-ended BamHI site of a K14 expression cassette generously
provided by E. Fuchs, Howard Hughes Medical Institute, Rockefeller University,
New York (Vasioukhin et al.,
1999). The K14 expression cassette
(Fig. 1A) contains a 2100 bp
AvaI fragment of the keratin 14 promoter/enhancer, a rabbit
ß-globin 5' untranslated region (UTR), together with an intronic
sequence upstream of a BamHI site, and the K14 3' UTR, followed
by a polyadenylation site 3' downstream of the BamHI site. The
transgene construct was excised from the pBabe vector as an
EcoRI/HindIII fragment, gel purified (Geneclean, Stratech
Scientific), further purified with an elutip column according to the
manufacturer's instructions (Schleicher and Schuell), and then resuspended at
a concentration of 5 µg/ml in sterile injection buffer (10 mM Tris-HCl, 0.1
mM EDTA, pH 7.4) for pronuclear injection.
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, as described previously
(Carroll et al., 1995).
Experimental treatments of mice
At the start of every experiment all the mice were 6-8 weeks old, and
therefore in the resting phase of the hair cycle
(Stenn and Paus, 2001). The
Nß-cateninER transgene was activated by topical application of
4-hydroxytamoxifen (4OHT; Sigma) to a clipped area of dorsal skin (4OHT was
dissolved in acetone; dose: 1 mg per mouse per day). Wild-type littermates
were used as controls. No sex-specific effects of 4OHT were observed. In some
experiments mice received an intraperitoneal injection of BrdU (0.1 mg/g body
weight) 1 hour prior to sacrifice.
Cell culture
Keratinocytes were isolated from transgenic and wild-type newborn mouse
skin and cultured at 32°C in a humidified atmosphere with 5%
CO2 in low calcium FAD medium containing 10% chelated FCS and a
cocktail of 0.5 µg/ml hydrocortisone, 5 µg/ml insulin,
10–10 M cholera toxin and 10 ng/ml EGF. The methods were
essentially as described previously
(Carroll et al., 1995;
Roper et al., 2001), except
that the skin trypsinisation procedure was carried out at 4°C and
disaggregation of keratinocytes was performed without stirring.
Phoenix and AM12 retroviral packaging lines and NIH 3T3 cells were cultured in DMEM containing 10% donor calf serum at 37°C in a humidified atmosphere with 5% CO2.
Retroviral infection and luciferase assays
Retroviral vectors encoding Nß-cateninER or GFP
(Lowell et al., 2000) were
packaged using a two step procedure involving transient transfection of
ecotropic Phoenix cells (Swift et al.,
1999) and infection of amphotropic AM12 cells with viral
supernatant from the Phoenix cultures (Zhu
and Watt, 1999; Watt et al.,
2004). NIH 3T3 cells were infected overnight at 32°C with
supernatant from transduced AM12 cells and selected with puromycin (2.0
µg/ml) to achieve close to 100% transduction efficiency.
The following Promega luciferase reporter constructs were used: pRL
(Renilla luciferase control), TOPFLASH (firefly luciferase) and FOPFLASH
(firefly luciferase) (van de Wetering et
al., 1997). Transient transfections of NIH 3T3 cells transduced
with Nß-cateninER or GFP retroviral vectors were performed using
Superfect Transfection Reagent (Qiagen). Cells were extracted using Passive
Lysis Buffer (Promega), enabling both firefly and Renilla luciferase
measurements to be performed on the same extracts. Luciferase assays were
performed according to the manufacturer's instructions using the
Dual-Luciferase Reporter Assay kit (Promega) on a BioOrbit 1251 luminometer.
All measurements were made in triplicate and corrected for transfection
efficiency.
Antibodies
Antibodies against the following antigens were used: ß-catenin (clone
15B8; Sigma), BrdU (Becton Dickinson), CCAAT displacement protein (CDP; a gift
from M. Busslinger) (Ellis et al.,
2001), cyclin D1 (Zymed), oestrogen receptor (MC-20; Santa Cruz
Biotechnology or HL-7) (Arnold and Watt,
2001), Erk2 (sc-1647; Santa Cruz Biotechnology), fatty acid
synthase (IBL), keratin 1 (Covance), keratin 10 (Covance), keratin 17 (a gift
from P. Coulombe) (McGowan and Coulombe,
1998), Lef1 (Upstate) and trichohyalin (AE15; a gift from
Tung-Tien Sun) (O'Guin et al.,
1992). Additionally, we generated the polyclonal antibody ERP2 by
injecting rabbits with a peptide (AHSLQTYYIPPEAEGFPNTI) corresponding to the C
terminus of the murine oestrogen receptor, which was previously used to
generate the HL-7 antibody (Arnold and
Watt, 2001). Goat anti-rabbit and anti-mouse Alexa 488 conjugated
IgG (Molecular Probes) and biotinylated goat anti-rabbit IgG (Vector
Laboratories) were used as secondary antibodies.
Histochemistry, immunohistochemistry and in situ hybridisation
Tissue samples for immunohistochemistry were either fixed overnight in
neutral buffered formalin and embedded in paraffin wax, or frozen, unfixed, in
OCT compound (Miles, Elkhart, USA) on a frozen isopentane surface (cooled with
liquid nitrogen). 5 µm sections were prepared and stained with Haematoxylin
and Eosin.
Immunohistochemistry for CDP, fatty acid synthase and the keratins was
performed on paraffin wax sections as described previously
(Niemann et al., 2002).
Briefly, antigen retrieval was performed by microwaving in 10 mM citrate
buffer (pH 6) for 20 minutes and non specific binding was blocked by
incubating the sections in 10% goat serum (Sigma) for 1 hour. Polyclonal
primary antibodies were diluted in PBS or TBS containing 1% BSA (ICN) and
detected using biotinylated secondary antibody (Vector Laboratories),
streptavidin complex with horseradish peroxidase (Dako) and DAB (Sigma). For
immunostaining of cyclinD1, trichohyalin and Lef1 the Mouse on Mouse kit
(Vector Laboratories) was used following the manufacturer's instructions.
Haematoxylin was used as a nuclear counterstain.
BrdU-labelled cells were detected in paraffin wax-embedded sections that
had been treated sequentially with HCl and trypsin
(Kim et al., 1997), using a
mouse monoclonal antibody to BrdU (Becton Dickinson) and Alexa 488-conjugated
goat anti-mouse IgG. To detect ER expression, 10 µm frozen sections were
fixed in 3% paraformaldehyde (Sigma) for 10 minutes, permeabilized with 0.2%
Triton X-100 (Sigma) for 5 minutes, incubated for 90 minutes in 10% goat serum
(Sigma) in PBS, then for 40 minutes with the ER antibodies and finally with
the Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes).
ß-catenin was visualised in the same way as ER, except that the Mouse on
Mouse kit (Vector Laboratories) was used. Nuclei were labelled with propidium
iodide and preparations were analysed on a Zeiss 510 confocal microscope.
Alkaline phosphatase activity was visualised in frozen sections with the
NBT-BCIP method (Filipe and Lake,
1990). Briefly, the sections were incubated in NTMT buffer (100 mM
Tris-HCl pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Triton X-100)
containing 4.5 µl/ml nitro blue tetrazoliun chloride (Roche) and 3.5
µl/ml 5-bromo-4-chloro-3-indolyl phosphate (Roche), counterstained with
Fast Red and mounted in Permount (Fischer Scientific).
In situ hybridisation was performed as described previously
(Poulsom et al., 1998), using
35S-labelled riboprobes to Sonic hedgehog (Shh) and Patched (Ptc),
kindly provided by B. Spencer-Dene (Revest
et al., 2001). Hybridisation with a ß-actin antisense probe
served as a positive control.
Western blotting
Primary mouse keratinocytes were cultured to confluence, then lysed in RIPA
buffer (50 mM Tris HCl pH7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl,
1 mM EGTA). Protein concentrations were determined using a BCA protein assay
kit (Pierce). Protein lysates (20 µg) were separated on a SDS-PAGE gel,
transferred to a PVDF membrane (Amersham) by electroblotting, blocked in 5%
non fat dried milk (Marvel, Cadbury), incubated with primary antibody and
visualised with appropriate horseradish peroxidase-coupled secondary
antibodies using enhanced chemiluminescence (ECL; Amersham).
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Nß-cateninER; Zhu and Watt,
1999). The C terminus of Nß-catenin was fused to the N
terminus of a form of the ligand binding domain of the oestrogen receptor
which is insensitive to endogenous oestrogen but responds to the synthetic
steroid 4-hydroxytamoxifen (4OHT)
(Littlewood et al., 1995;
Gandarillas and Watt, 1997;
Arnold and Watt, 2001).
ß-cateninER chimeras have previously been shown to retain
ß-catenin activity (Weng et al.,
2002; Kolligs et al.,
2002; Van Mater et al.,
2003). To establish that this was also the case for our construct
we introduced Nß-cateninER into a retroviral vector and transduced
NIH 3T3 cells (Fig. 1B). As
predicted, in the absence of 4OHT the chimeric protein was expressed and
accumulated primarily in the cytoplasm, where it was detected with an antibody
to the mutant oestrogen receptor (Fig.
1B, left hand panel). When cells were treated with 4OHT the
cytoplasmic staining was reduced and the chimera was localised predominantly
in the nucleus (Fig. 1B, right
hand panel). This change in the expression pattern was expected, since in the
absence of 4OHT the ER is complexed with heat shock proteins in the cytoplasm
(Littlewood et al., 1995).
To examine whether Nß-cateninER could activate transcription of
Lef/Tcf target genes we retrovirally transduced 3T3 cells with the
Nß-cateninER construct or with GFP
(Lowell et al., 2000) and we
transfected them with a luciferase construct containing an enhancer with
multiple Lef1/Tcf-binding sequences (TOP)
(van de Wetering et al.,
1997). As a negative control we used a reporter construct with
mutated Lef1/Tcf binding sites (FOP) (van
de Wetering et al., 1997). 4OHT treatment led to a three-fold
increase in luciferase activity of the TOP construct in cells expressing
Nß-cateninER but had no effect on the FOP construct
(Fig. 1C). 4OHT had no effect
on TOP or FOP activity in cells transduced with GFP
(Fig. 1C).
Having established that Nß-cateninER was able to inducibly
activate transcription in cultured cells we generated transgenic mice in which
the construct was expressed under the control of the keratin 14 (K14) promoter
(Fig. 1A). The K14 promoter is
active in all the basal cells of interfollicular epidermis and along the
length of the hair follicle outer root sheath
(Vassar et al., 1989;
Byrne et al., 1994;
Wang et al., 1997). Four
K14Nß-cateninER founder lines were chosen for analysis, based on
the range in transgene copy number that they represented. Line 3953 has a
single copy of the transgene; line D2 has 12 copies; line C5 has 18 copies and
line D4 has 21 copies. None of the mice exhibited any phenotype in the absence
of 4OHT.
Expression of the transgene was examined by immunofluorescence labelling of
skin sections (Fig. 1D) and by
western blotting of cultured primary mouse keratinocytes
(Fig. 1E), using antibodies to
the oestrogen receptor. Epidermis from transgene-negative mice did not express
any protein that could be detected with the anti-ER antibodies
(Fig. 1D,E). In transgenic
mouse skin that had not been treated with 4OHT, cytoplasmic staining for ER
was detected in the basal layer of the interfollicular epidermis and along the
outer root sheath of the hair follicle with no obvious cell to cell variation
in levels (Fig. 1D, middle
panel). In 4OHT-treated epidermis, nuclear staining was evident in all
transgene-positive cells, although it was more intense in the hair follicles
than in the interfollicular epidermis (Fig.
1D, right panel and data not shown). Nuclear staining for ER and
ß-catenin was similar (data not shown). The level of
Nß-catenin ER protein correlated with transgene copy number, with
line 3953 keratinocytes expressing the lowest level of the protein and line D4
keratinocytes expressing the highest level
(Fig. 1E and data not
shown).
Consequences of activating ß-catenin for different lengths of time
In pilot experiments we applied 4OHT topically to the back skin of
transgenic and wild-type mice daily and assessed whether there was any
macroscopic change in phenotype (Fig.
1F and data not shown). The mice were 6 weeks old at the start of
treatment and were thus in the resting (telogen) phase of the hair cycle. As a
consequence, clipped wild-type animals did not regrow hair, whether treated
with 4OHT (Fig. 1F, left panel)
or with acetone vehicle (data not shown). The D2 line
(Fig. 1F, right panel) showed
complete hair regrowth within 14 days. The 3953 line showed patchy hair
regrowth (data not shown) and as the histological changes were similar to D2,
only less pronounced, we did not study this line further. After 9 days of 4OHT
treatment the skin of D4 transgenic mice was very dark and wrinkled, and
although new hair growth was stimulated the hairs failed to fully elongate
(data not shown). The C5 line showed the same gross phenotype as line D4,
although the onset of the 4OHT-induced changes was slightly later (data not
shown).
We next performed a time course experiment in which we analysed sections of
back skin from D2, D4 and littermate control mice at intervals for up to 14
days of daily 4OHT treatment (Fig.
2). No effects of 4OHT on the back skin of transgene-negative mice
were seen (Arnold and Watt,
2001) (data not shown). 4OHT did not cause any changes in the
histology of the interfollicular epidermis in D2 mice, while in the D4 line we
observed occasional patches of hyperproliferative epidermis (e.g.
Fig. 2, day 9).
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In D4 transgenics an elongation of the hair follicles was already visible 1 day after the beginning of 4OHT treatment (Fig. 2). By day 3, epithelial outgrowths were developing from the sebaceous glands (arrow in Fig. 2) and from the permanent portion of the outer root sheath below the sebaceous glands. Between day 5 and day 9 the follicles became progressively thicker, with the outgrowths becoming larger. In contrast to the hair follicles of D2 mice the D4 follicles tended not to produce normal hair shafts and this probably accounts for the abnormal macroscopic appearance of the pelage.
The D4 mice had to be sacrificed by day 11 and the C5 mice by day 17
because they developed thickening of the lips and tongue which affected their
ability to feed. The D2 line could, however, be maintained indefinitely with
4OHT treatment. By day 21 the outgrowths from D2 hair follicles had become
very pronounced and small cyst-like structures, corresponding to empty hair
canals, had appeared (arrowhead, Fig.
2). The histology of each D2 follicle from day 21 onwards
resembled a trichofolliculoma, a hair follicle tumour that has previously been
reported to be induced by constitutive overexpression of stabilised
ß-catenin (Gat et al.,
1998). At day 28, the follicles of D2 epidermis were entering
catagen (regression phase) but the tumours were still enlarging and thus the
epithelial outgrowths accounted for most of the mass of the follicles.
ß-catenin induces de novo hair follicle formation in postnatal interfollicular epidermis
In addition to the changes in existing hair follicles
(Fig. 2), de novo hair follicle
formation was observed in D2 and D4 transgenics
(Fig. 3). This was particularly
striking in paw skin. On the dorsal surface of the paw the density of
follicles was higher in transgenics treated with 4OHT for 7 days
(Fig. 3B,F) than in littermate
controls (Fig. 3A,E). In the
transgenic skin many downgrowths of interfollicular epidermis into the
underlying dermis were observed, which had the appearance of rudimentary hair
follicles (arrows in Fig. 3F).
On the dorsal region of the digits of D4 mice the interfollicular epidermis
was hyperproliferative, with an excess of cornified material extending into
the neck of pre-existing hair follicles
(Fig. 3F). The induction of new
rudimentary and mature hair follicles was not confined to the paws, since new
hair follicle formation was observed in dorsal skin of D2 and D4 mice
(Fig. 3G, and data not
shown).
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). Similar alterations at the dermal-epidermal boundary were
seen also in the internal surface of the ear of transgenic mice (data not
shown).
At the base of wild-type follicles the epithelial cells ensheath
specialised mesenchymal cells, known as dermal papilla cells, which express
high levels of alkaline phosphatase (Van
Mater et al., 2003) (Fig.
3G,H,I). The mesenchyme adjacent to the epithelial projections,
both from existing follicles and the interfollicular epidermis, had high
alkaline phosphatase activity (Fig.
3G,H,J). It thus appears that keratinocytes expressing high levels
of ß-catenin are able to induce dermal papillae.
One of the earliest features of de novo hair follicle formation is
upregulation of Sonic hedgehog (Shh) and its receptor Patched (Ptc)
(St-Jacques et al., 1998;
Chiang et al., 1999), which
are required for the maturation of the hair germ at the base of the follicle.
We therefore used in situ hybridisation to examine whether Shh and Ptc
expression was increased in response to activation of Nß-cateninER
(Fig. 4). In wild-type anagen
hair follicles expression of Shh was confined to a group of matrix cells on
one side of the bulb and Ptc was expressed in all the adjacent cells
(Fig. 4A-D, arrows)
(Oro and Higgins, 2003). In
4OHT-treated D2 (Fig. 4E-H) and
D4 (Fig. 4I-L) back skin, Shh
and Ptc were upregulated in the epithelial outgrowths from existing hair
follicles. In D4 back skin follicles Shh expression was no longer asymmetric
but rather was observed on both sides of the hair bulb
(Fig. 4I,J). Shh and Ptc were
also expressed in the downgrowths of interfollicular epidermis on the dorsal
surface of the paws of D4 mice (Fig.
4M-P). The induction of Shh and Ptc in all the observed epithelial
outgrowths supports the conclusion that they are rudimentary hair
follicles.
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).
By 5 days of 4OHT treatment there was increased proliferation in the epidermis
of transgenic mice, but no effect on proliferation in the epidermis of
wild-type littermate controls (Fig.
5 and data not shown). In D2 and D4 mice the increase in
BrdU-positive cells occurred along the ORS, at the base of the hair follicles
and in the epithelial outgrowths from the interfollicular epidermis and
existing hair follicles, but not in the interfollicular epidermis itself
(Fig. 5B-D). In D4 mice the
increase in BrdU incorporation was more pronounced than in D2 mice and
BrdU-positive cells were often found in more than one cell layer, reflecting
the thickening of the ORS (Fig.
5B). Except in the epithelial downgrowths there was no increase in
BrdU incorporation in the phenotypically normal interfollicular epidermis of
treated transgenics (Fig. 5B,C) compared to wild-type mice (Fig.
5A). The expression of cyclin D1 mirrored BrdU incorporation, with
increased labelling in the ORS, the epithelial outgrowths from the hair
follicles and interfollicular epidermis, and at the base of follicles
(Fig. 5E,F and data not
shown).
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Nß-catenin was inducing new hair follicle
formation. For further confirmation, we examined expression of a range of
markers of hair follicle differentiation
(Fig. 6). In wild-type
follicles keratin 17 is expressed along the outer root sheath
(Fig. 6A)
(McGowan and Coulombe, 1998),
while the transcriptional repressor CCAAT displacement protein (CDP),
essential for normal hair follicle inner root sheath formation, is confined to
the bulb (Fig. 6B)
(Ellis et al., 2001).
Trichohyalin is a terminal differentiation marker expressed in cells that are
producing the hair shaft (O'Guin et al.,
1992) (Fig. 6C) and
Lef1 is localised in the matrix (Fig.
6D) (DasGupta and Fuchs,
1999). In 4OHT-treated D2 transgenics expression of keratin 17
(Fig. 6E), CDP
(Fig. 6F) and Lef1
(Fig. 6H) was observed in the
outgrowths from the hair follicles, but the outgrowths did not express
trichohyalin (Fig. 6G). In D4
mice all four markers were expressed in the new follicles forming from
interfollicular epidermis and pre-existing follicles
(Fig. 6I-L). These results
support the conclusion from the histological examination of the skin (Figs
2,
3) that ß-catenin promotes
hair lineage differentiation. The increase in proliferation
(Fig. 5) coincided with, rather
than preceded, the ectopic expression of hair differentiation markers
(Fig. 6).
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)
(Fig. 7A,B) we frequently
observed duplication of the sebaceous glands in D2 and C5 epidermis during the
first 15 days of 4OHT treatment (data not shown and
Fig. 7C). At later time points
there was loss of morphologically identifiable sebaceous glands in D2 skin and
sebocytes were found only in small clusters in a scattered distribution in the
trichofolliculomas (Fig. 7D). In the D4 line sebaceous gland differentiation was lost within 9 days
(Fig. 7E). This suggests that
sustained activation of ß-catenin signalling promotes differentiation
along the hair lineages at the expense of sebaceous differentiation.
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In D2 and D4 mice a single application of 4OHT induced the same changes as seven daily treatments (Fig. 8B-D; compare Fig. 2). One dose of 4OHT was sufficient to induce anagen and epithelial outgrowths from hair follicles in D2 mice (Fig. 8B; compare Fig. 2). One dose of 4OHT induced thickening of the hair follicles (Fig. 8C) and epithelial outgrowths from the outer root sheath (Fig. 8C) and interfollicular epidermis (Fig. 8D) in D4 skin (compare with Fig. 2). Similarly, mice treated for 5 days with 4OHT and then examined after 5 or 10 days (Fig. 8A) were phenotypically indistinguishable from mice treated continuously for 10 or 15 days (data not shown).
In contrast to the ability of transient ß-catenin activation to induce anagen and de novo follicle formation, continuous ß-catenin expression was required to maintain hair follicle tumours (Fig. 8E-G). The tumours observed after 28 days of continuous ß-catenin activation (Fig. 8E) started to regress after 4OHT treatment had ceased (Fig. 8F). The skin did not return completely to normal, however, as the hair follicles retained small epithelial outgrowths and cysts as late as 106 days after 4OHT treatment had stopped (Fig. 8G). Remarkably, in ear skin the tumours continued to expand for about 2 weeks in the absence of 4OHT before regression began (data not shown). While the tumours induced by continuous ß-catenin activation were positive for cyclin D1 (Fig. 8H), Lef1 (Fig. 8I) and keratin 17 (Fig. 8J), during regression Lef1 and cyclin D1 expression were lost (Fig. 8L and data not shown). The residual tumour masses remained positive for keratin 17 (Fig. 8M) and negative for the interfollicular epidermal marker keratin 1 (Fig. 8K). Whereas the tumours showed strong staining for nuclear ß-catenin (Fig. 8N,O) this was lost when the tumours regressed (Fig. 8P,Q).
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Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Byrne et al., 1994;
Wang et al., 1997). It has
been used to manipulate ß-catenin signalling in several different
transgenic models, thereby demonstrating that ß-catenin plays a key role
in epidermal lineage commitment (e.g. Gat
et al., 1998; Merrill et al.,
2001; Niemann et al.,
2002; DasGupta et al.,
2002). However, the endogenous promoter is first active at E9.5,
before the hair placodes form (Byrne et
al., 1994), and so it is possible that transgene expression during
embryonic development is necessary to render the epidermis responsive to
changes in ß-catenin signalling in postnatal life. In order to establish
whether the epidermis is truly capable of undergoing lineage conversion in
adulthood we generated an inducible form of ß-catenin under the control
of the K14 promoter. This allowed us to activate ß-catenin signalling
exclusively in postnatal epidermis and to investigate the effects of
activating ß-catenin for different lengths of time. A similar approach
has recently been described by Van Mater et al.
(Van Mater et al., 2003) who
found, as we did, that transient ß-catenin activation is sufficient to
induce hair follicles to enter the growth phase (anagen) of the hair cycle. In
addition we show that ß-catenin induces de novo hair follicle formation
in interfollicular epidermis and can cause either duplication of the sebaceous
glands or inhibition of sebocyte differentiation depending on the level of
activation. These results reveal a remarkable plasticity in the
differentiation potential of adult epidermis.
In all of the K14Nß-catenin lines the first effect of applying
4OHT to skin with telogen (resting) hair follicles was to induce anagen. In D2
mice the anagen was essentially normal and the coat of fur that grew could not
be distinguished macroscopically from a wild-type pelage. However, in D4 mice,
which have a higher transgene copy number and express a higher level of
Nß-catenin, the degree of proliferation induced by 4OHT was
greater than in normal anagen with the result that the follicles became
abnormally thickened and failed to produce normal hair shafts. Anagen was
induced as effectively by transient as by continuous activation of
ß-catenin (see also Van Mater et al.,
2003). Conversely, anagen did not continue indefinitely, since
follicles in epidermis treated with 4OHT for 28 days were shorter than
follicles treated for 21 days, demonstrating that additional factors are
required for maintenance of hair growth
(Fuchs et al., 2001).
Shh is a target gene of ß-catenin in the epidermis
(Gat et al., 1998;
Huelsken et al., 2001) and was
induced by activation of Nß-cateninER. Shh is expressed in the
bulb of growing wild-type follicles and is known to initiate anagen
(Oro et al., 1997;
St-Jacques et al., 1998;
Sato et al., 1999;
Wang et al., 1997;
Callahan and Oro, 2001;
Stenn and Paus, 2001). It
therefore seems likely that the reason for the entry of transgenic follicles
into anagen was that Shh was induced by Nß-cateninER. Levels of
Shh and its receptor Ptc, itself a Shh target
(Freeman, 2000), were higher
in D4 than in D2 epidermis and whereas in wild-type and D2 follicles Shh
expression was asymmetric, in D4 follicles expression was observed on both
sides of the hair bulb. Thus, aberrant Shh levels and localisation may
contribute to the abnormalities in D4 anagen follicles.
De novo follicle formation was a striking phenotype of
K14Nß-cateninER mice and established that ß-catenin can
induce new follicles even when expression is restricted to postnatal epidermis
(cf. Gat et al., 1998).
Rudimentary follicles developed as epithelial outgrowths from the permanent
portion of the outer root sheath, from sebaceous glands and from
interfollicular epidermis. Such outgrowths also formed on the ventral surface
of the paw and in the internal surface of the ear, regions normally devoid of
hair follicles. Dermal cells adjacent to the outgrowths had high alkaline
phosphatase activity, a marker of dermal papilla cells. At later times the
base of the new follicles was seen enveloping a dermal condensate that was
morphologically indistinguishable from a dermal papilla (data not shown).
All of the outgrowths expressed Shh, Ptc, K17, CDP and Lef1, markers of
hair follicle formation (Millar,
2002; McGowan and Coulombe,
1998; Ellis et al.,
2001; Zhou et al.,
1995). Whether the outgrowths progressed to form complete hair
follicles with hair shafts and trichohyalin expression depended on the
duration of ß-catenin activation, complete follicles appearing from 14
days onwards. The level of ß-catenin appeared to determine the number of
new follicles developing from interfollicular epidermis: in the D4 transgenic
line there were more new follicles and the initial outgrowths appeared in a
shorter time than in the D2 line.
The formation of hair follicles during embryogenesis depends on a series of
signals that are exchanged between the epidermis and the underlying dermis
(Hardy, 1992;
Millar, 2002). In the embryo
the initiating signal comes from the dermis and involves activation of Wnt
signalling (Kishimoto et al.,
2000); the response in the overlying epithelium also involves
activation of ß-catenin. In K14Nß-cateninER skin the dermal
signal is not required and activation of ß-catenin signalling in the
epidermis leads to organisation of a dermal papilla
(Fig. 3G-J). In each location
where new follicles formed in K14Nß-cateninER epidermis there was
induction of Shh, Ptc and Lef1. Just as Shh drives anagen, Shh is downstream
of Wnt signalling in hair follicle development
(Fuchs et al., 2001;
Millar, 2002): in mice lacking
Shh hair follicle formation is initiated and the dermal condensate is formed
but mature hair follicles fail to develop
(St-Jacques et al., 1998;
Chiang et al., 1999). Lef1 is
a known transcriptional target of ß-catenin
(Filali et al., 2002),
required for normal hair follicle formation
(van Genderen et al., 1994;
Zhou et al., 1995;
Kratochwil et al., 1996).
Although during normal hair placode formation expression of Lef1 is regulated
by Noggin, produced by dermal cells
(Jamora et al., 2003), in our
system Lef1 upregulation is independent of a pre-existing dermal signal.
The effects of prolonged activation of Nß-cateninER were not
confined to hair follicle differentiation. While differentiation within the
interfollicular epidermis was largely normal there were also some areas of
hyperproliferation, leaving open the question of whether ß-catenin can
expand the size of the IFE stem cell pool
(Zhu et al., 1999;
Reya et al., 2003). The
effects on sebocyte differentiation were even more puzzling, because there was
initial duplication of sebaceous glands, but thereafter sebocytes were lost;
in D4 epidermis no sebocytes were detectable by 9 days of 4OHT treatment.
c-Myc is induced by ß-catenin (He et
al., 1998) and direct activation of c-Myc in adult epidermis
stimulates sebocyte differentiation in both the sebaceous glands and
interfollicular epidermis (Arnold and Watt,
2001; Braun et al.,
2003). However, when ß-catenin signalling is blocked with a
NLef1 transgene there is ectopic sebocyte differentiation and sebocyte
tumours appear (Niemann et al.,
2002; Braun et al.,
2003). One interpretation of these results is that sebocyte
differentiation is promoted by intermediate levels and duration of
ß-catenin signalling and blocked by higher levels and duration. The
phenotype of epidermis expressing NLef1 demonstrates that sebocyte
differentiation can be promoted independently of ß-catenin activation,
presumably through the ability of NLef1 to interact with other pathways
(Labbe et al., 2000).
Just as ß-catenin levels regulate lineage choice in the epidermis so
they also influence the types of tumour that develop when signalling is
deregulated (Owens and Watt,
2003). High ß-catenin expression results in hair follicle
type tumours (Gat et al.,
1998; Chan et al.,
1999) whereas inhibition of ß-catenin signalling with
NLef1 leads to the formation of sebaceous tumours at high frequency
(Niemann et al., 2002;
Niemann et al., 2003). The
tumours induced by sustained activation of Nß-cateninER were
highly differentiated and resembled human trichofolliculomas, consistent with
the earlier observations of Gat et al.
(Gat et al., 1998) and Chan et
al. (Chan et al., 1999). Every
follicle in which Nß-catenin was activated for 21 days developed
into a tumour. However, once ß-catenin activation ceased all the tumours
regressed, and by three months what remained were remnants of the tumour mass
that mainly consisted of empty hair shafts. Thus continued ß-catenin
activation was needed to maintain the tumours, and they could not grow
autonomously. One reason may be that ß-catenin induces p53, which serves
to limit growth (Damalas et al.,
2001). Additional oncogenic changes, such as mutation of p53 or
Ras, would be necessary to convert the trichofolliculomas into malignant
tumours capable of autonomous growth (Hahn
and Weinberg, 2002;
Perez-Losada and Balmain,
2003; Owens and Watt,
2003).
Considerable debate surrounds the degree of plasticity of adult stem cells
(Alison et al., 2003;
Goodell, 2003). While the
current consensus is that the ability of adult stem cells to undergo
conversion into unrelated cell types is limited (e.g.
Wagers et al., 2002),
interconversion between epidermal lineages can be readily induced. The present
studies show that ß-catenin can induce de novo hair follicle formation
from existing follicles, interfollicular epidermis and sebaceous glands, and
it has previously been reported that c-Myc induces sebocyte differentiation in
interfollicular epidermis (Braun et al.,
2003). Thus all regions of the epidermis are competent to undergo
lineage conversion. We and others have argued that there are distinct stem
cell pools in IFE, sebaceous glands and hair follicles (reviewed by
Niemann and Watt, 2002). The
challenge now is to find out whether only stem cells are capable of undergoing
reprogramming or whether plasticity is also retained in committed progenitors
(Braun et al., 2003).
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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Present address: Institute of Cell and Molecular Science, Barts and the
London Queen Mary's School of Medicine and Dentistry, Centre for Cutaneous
Research, 2 Newmark Street, London E1 2AT, UK
|
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