1 Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44
Lincoln's Inn Fields, London WC2A 3PX, UK
2 Department of Human Genetics, The Bartholin Building, University of Aarhus,
DK-8000 Aarhus C, Denmark
3 The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609-1500, USA
* Author for correspondence (e-mail: fiona.watt{at}cancer.org.uk)
Accepted 2 July 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Myc, Lef1, Hair follicle, Hair cycle, Stem cells, Epidermis, Sebocytes, Differentiation, Label-retaining cells, ß-catenin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Epidermal stem cells can be distinguished from their non-stem progeny
because they not only lack expression of markers of terminal differentiation,
but also have distinct proliferative characteristics. In particular, they
divide infrequently in undamaged, steady-state epidermis
(Potten and Morris, 1988;
Cotsarelis et al., 1990
), yet
have the ability to undergo considerable proliferation in response to stimuli
such as wounding. These properties form the basis of two different approaches
to the identification of stem cells. The first is to give neonatal mice
repeated injections of 3H-thymidine or 5-bromo-2
'-deoxyuridine (BrdU) to label all the dividing cells in the epidermis
at a time of rapid tissue expansion and then to identify those cells that do
not divide subsequently and thus retain the label into adulthood
(label-retaining cells, LRC) (Bickenbach,
1981
; Morris et al.,
1985
; Bickenbach et al.,
1986
; Cotsarelis et al.,
1990
; Bickenbach and Chism,
1998
; Morris and Potten,
1999
). The second is to place epidermal cells in culture and
perform clonal analysis: stem cells form large, self-renewing clones, whereas
their non-stem daughters (known as transit amplifying cells or committed
progenitors) (Potten and Morris,
1988
; Watt, 1998
)
form abortive clones, because they divide a small number of times, then
withdraw from the cell cycle and undergo terminal differentiation
(Barrandon and Green, 1987
;
Jones and Watt, 1993
;
Morris and Potten, 1994
). From
experiments in which LRC and clonogenic cells have been compared directly
there is evidence that stem cells do indeed share label-retaining and
clone-forming ability (Morris and Potten,
1994
; Oshima et al.,
2001
).
Postnatal hair follicles undergo repeated rounds of growth (anagen),
regression (catagen) and quiescence (telogen)
(Hardy, 1992;
Stenn and Paus, 2001
). During
catagen, the keratinocytes in the lower (cycling) region of the follicle are
destroyed, leaving only the non-cycling upper one-third of the hair follicle
intact, along with the mesenchymal cells of the dermal papilla. In mammalian
hair follicles, stem cells are concentrated in the non-cycling region of the
follicle in a specialised region of the outer root sheath
(Cotsarelis et al., 1990
;
Lavker et al., 1993
;
Lyle et al., 1998
;
Morris and Potten, 1999
;
Akiyama et al., 2000
;
Taylor et al., 2000
;
Fuchs et al., 2001
;
Oshima et al., 2001
). This
region is defined by the point of insertion of the arrector pili muscle
(Cotsarelis et al., 1990
) and
is known as the bulge.
The availability of promoters that target transgene expression to the
epidermis has provided a wealth of information about the molecules that
regulate epidermal self-renewal and differentiation
(Fuchs and Raghavan, 2002;
Niemann and Watt, 2002
).
However, the number of label-retaining cells is so small that it has proved
hard to examine the effects of the transgenes on the epidermal stem cell
compartment directly. One approach is to view LRC by preparing serial
reconstructions of individual hair follicles
(Morris and Potten, 1999
);
however, this is an extremely laborious technique that is not amenable to the
screening of large numbers of follicles or large areas of IFE. In human
epidermis, visualisation of the stem cell compartment has been facilitated by
preparation of wholemounts, in which the epidermis is separated from the
underlying dermis as an intact sheet, then labelled with antibodies to markers
of proliferation or differentiation
(Jensen et al., 1999
). In the
present report we describe the development of a similar whole-mount labelling
technique for mouse epidermis.
We have used whole-mount labelling to determine the number and location of LRC and as a screen for molecular markers of LRC. We have also examined what happens to LRC during the normal hair growth cycle and when epidermal growth and differentiation are disturbed. By simultaneously visualising all the LRC within large numbers of HF and large areas of the IFE we have clarified some important aspects of their biology.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BrdU labelling
To generate label-retaining cells (LRC), the protocol described by
Bickenbach and colleagues (Bickenbach et
al., 1986; Bickenbach and
Chism, 1998
) was used. Ten-day-old mice were injected with 50
mg/kg bodyweight 5-bromo-2'-deoxyuridine (BrdU; 20 µl of 12.5 mg/ml
BrdU) every 12 hours for a total of four injections to label mitotic cells. By
injecting neonatal mice we avoided the teratogenic effects of BrdU
(Shah and MacKay, 1978
;
Bannigan et al., 1990
). The
mice were healthy and the only evidence that they were affected by BrdU was a
transient loss of dorsal hair prior to the first postnatal hair cycle.
An extremely high percentage of keratinocytes incorporate the BrdU label because the epidermis is hyperproliferative in 10-day-old mice. Normally, mice were maintained for a minimum of 70 days after the final BrdU injection in order to detect LRC. In some experiments, the localisation of cells containing the BrdU label was assessed after a chase period of less than 70 days. To achieve short-term labelling of cells undergoing DNA synthesis, adult mice received an intraperitoneal injection of 100 mg/kg body weight 5-bromo-2'-deoxyuridine (BrdU; Sigma) 1 hour prior to sacrifice.
Preparation of wholemounts
The method described to prepare human epidermal wholemounts
(Jensen et al., 1999) was
modified for mouse tissue. To prepare wholemounts of mouse tail epidermis, a
scalpel was used to slit the tail lengthways. Skin was peeled from the tail,
cut into pieces (0.5x0.5 cm2) and incubated in 5 mM EDTA in
PBS at 37°C for four hours. Forceps were used to gently peel the intact
sheet of epidermis away from the dermis and the epidermal tissue was fixed in
4% formal saline (Sigma) for 2 hours at room temperature. Fixed epidermal
sheets were stored in PBS containing 0.2% sodium azide at 4°C for up to 8
weeks prior to labelling.
To prepare wholemounts from mouse back skin, the skin surface was shaved with electric clippers and treated topically with hair removal cream (ImmacTM) for 5 minutes. Remaining hair was scraped from the dorsal surface and the skin was removed from the mouse. Fat and most of the connective tissue were separated using scissors and the skin was cut into pieces (0.5x0.5 cm2). Skin was incubated in 0.25% trypsin at 4°C for 24 to 48 hours; the epidermis was removed as soon as it could be separated from the dermis as an intact sheet. Fixation and storage conditions were the same as described above.
Antibodies
Rat monoclonal antibodies were used to detect BrdU (Oxford Biotechnology),
6 integrin (Serotec) and ß1 integrin (MB 1.2, kindly provided by
B. Chan) (Von Ballestrem et al.,
1996
). Polyclonal rabbit antiserum was used to detect keratin 6
(Babco), keratin 10 (Babco), keratin 14 (Babco), keratin 17 (kind gift of P.
Coulombe) (McGowan and Coulombe,
1998
), Ki67 (Novacastra), CCAAT displacement protein (CDP) (kindly
provided by M. Busslinger) (Ellis et al.,
2001
) and NG2 chondroitin sulphate proteoglycan (Chemicon). Mouse
monoclonal antibodies were used to detect BrdU (Becton Dickinson) and keratin
15 (kindly provided by I. Leigh) (Waseem
et al., 1999
). Secondary antibodies were conjugated to AlexaFluor
488 or AlexaFluor 594 (Molecular Probes).
Histological analysis and immunolabelling of tissue sections
Mice were killed with CO2. Skin sections were harvested and
either fixed overnight in 10% neutral-buffered formalin or frozen, unfixed, in
OCT compound (Miles) on a frozen isopentane surface (cooled with liquid
nitrogen). Formalin-fixed tissue was transferred to 70% ethanol, embedded in
paraffin wax and sectioned at 5 µm. Paraffin wax and frozen skin sections
were stained with Haematoxylin and Eosin (H&E) and then examined
microscopically. Lipid-containing cells, including sebocytes, were detected by
performing Oil Red O staining on frozen sections essentially as described
previously (Catalano and Lillie,
1975). To assess the hair cycle, skin sections were examined and
the relative percent of follicles in anagen, catagen or telogen was estimated
using criteria described elsewhere
(Sundberg et al., 1996
;
Sundberg et al., 1997
;
Sundberg and King, 2000
).
Double-immunolabelling was used to detect keratin 14 and BrdU in paraffin wax-embedded sections of mouse tail skin. Formalin-fixed sections were deparaffinised in xylene and rehydrated in graded alcohols. Tissue sections were microwaved in 10 mM sodium citrate (pH 6.0) for 3 minutes, incubated for another 15 minutes in the hot solution and rinsed in Automation Buffer (Biomedia, Foster City, CA). Sections were incubated in 2 M HCl at 37°C, washed in borate buffer, and digested in 0.01% trypsin in 0.05 M Tris for 3 minutes at 37°C. After blocking in 10% goat serum for 20 minutes, sections were incubated for 1 hour at room temperature with mouse BrdU antisera (Becton Dickinson; 1:25) and keratin 14 antisera (1:10000) in 1% bovine serum albumin.
Immunolabelling, staining and confocal microscopy of epidermal
sheets
Epidermal sheets were blocked and permeabilised by incubation in PB buffer
for 30 minutes (Jensen et al.,
1999). PB buffer consists of 0.5% skim milk powder, 0.25% fish
skin gelatin (Sigma) and 0.5% Triton X-100 in TBS (0.9% NaCl, 20 mM HEPES, pH
7.2). Primary antibodies were diluted in PB buffer and tissue was incubated
overnight at room temperature with gentle agitation. Epidermal wholemounts
were then washed for at least 4 hours in PBS containing 0.2% Tween 20,
changing the buffer several times. Incubation with secondary antibodies was
performed in the same way. Samples were rinsed in distilled water and mounted
in Gelvatol (Monsanto, St Louis, MO) containing 0.5%
1,4-Diazabicyclo[2.2.2]octane (DABCO) (Sigma). To detect BrdU-labelled cells,
after permeabilisation and prior to incubation with the anti-BrdU antibody,
epidermal sheets were incubated for 20-30 minutes in 2M HCl at 37°C.
Apoptotic cells were detected in epidermal sheets by using the
DeadEndTM Fluorometric TUNEL System (Promega). To stain
lipids present in sebaceous glands, epidermal sheets were incubated in Nile
Red (0.1 µg/ml in PBS; Sigma) for 30 minutes at room temperature, washed
several times in PBS and mounted.
In some experiments, a dissecting microscope was used to isolate individual hair follicles from labelled epidermal sheets. The epidermis was placed in PBS with the basal layer facing upwards. Fine forceps were used to grasp a hair follicle at the infundibulum and the follicle was pulled gently until it separated from the epidermal sheet. Hair follicles were mounted on a slide in Gelvatol with 0.5% DABCO.
Images were acquired using a Zeiss 510 confocal microscope. Approximately 30 optical sections of each epidermal sheet were captured with a typical increment of 1-3 µm. Scans are presented as z-projections. Objectives used were Zeiss 10/NA 0.45, Zeiss 20/NA 0.75 and Zeiss 40/NA 1.2. The samples were scanned from the dermal side towards the epidermal surface to a total thickness of 40-80 µm, which encompassed the epidermis from the hair follicle bulb to the basal layer of interfollicular epidermis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In pilot studies, it proved difficult to produce consistently high-quality
wholemounts from mouse dorsal skin because of the high density of hair
follicles and thin interfollicular epidermis. By contrast, mouse tail skin was
amenable to the production of epidermal wholemounts in which the pilosebaceous
units remained intact (Fig.
1A). Mouse tail hair follicles are known to be arranged in
parallel rows, the follicles being grouped in sets of three (triplets)
(Schweizer and Marks, 1977a;
Schweizer and Marks, 1977b
),
and this organisation was clearly visible in the wholemounts
(Fig.1A-D). Wholemounts were
labelled with an antibody to keratin 14 (K14) in order to visualise all the
cells in the basal layer of the IFE, the outer root sheath (ORS) of the hair
follicle and the outer layer of the sebaceous gland
(Fig. 1A-D).
|
LRC were generated using a previously reported protocol
(Bickenbach et al., 1986;
Bickenbach and Chism, 1998
) in
which 10-day-old mice received an injection of BrdU every 12 hours for a total
of four injections. BrdU initially labels the majority of cells in the
epidermis that are capable of proliferating, including the stem cells. During
the chase period, the label is lost from dividing cells, so that only
infrequently cycling cells, the putative stem cells, retain the label. To
assess the efficiency of BrdU labelling, mice were killed 2 days after the
final injection of BrdU: the majority of cells within the basal layer of the
IFE, hair follicle outer root sheath and outer layer of the sebaceous glands
contained the BrdU label (Fig.
1B). From 24 days post-labelling (anagen of the first post-natal
hair cycle), the pattern of incorporation remained relatively constant. Some
keratinocytes in both the HF and IFE still retained the BrdU label after a 140
day chase (Fig. 1C), which
demonstrated that long-term labelling of infrequently cycling cells could be
achieved using this protocol.
As previous studies of LRC have focused on mouse dorsal skin
(Cotsarelis et al., 1990;
Taylor et al., 2000
) we
compared the number and location of LRC in hair follicles on the dorsal and
tail skin. We had some success in preparing dorsal epidermal wholemounts by
harvesting skin at the telogen phase of the hair cycle (see also
Sundberg et al., 1993
;
Zhang et al., 2001
).
Wholemounts of tail and dorsal epidermis were prepared from 42-day-old mice
that were in telogen (31 days post-BrdU injection;
Fig. 1D,E). In both types of
epidermis, LRC were distributed as single cells within the IFE and clustered
in the permanent portion of the hair follicle
(Fig. 1D,E). The main
difference between the two types of epidermis was that the number of LRC per
hair follicle was greater in tail (range 10-79 LRC; see
Table 1) than in dorsal (range
0-15 LRC; n=10) follicles (Fig.
1D,E). This difference reflects the fact that hair follicles in
tail epidermis are much larger than dorsal follicles (compare
Fig. 1D with 1E, same
magnification).
|
The LRC were present in all follicles, irrespective of whether a distinct
bulge was visible (Fig. 1C,F).
This protuberance of the outer root sheath may be caused by the presence of
the club hair (Morris and Potten,
1994; Narisawa and Kohda,
1996
; Stenn and Paus,
2001
). Whereas the visible bulge lies on one side of the follicle
(Cotsarelis et al., 1990
;
Morris and Potten, 1994
), the
LRC were usually distributed symmetrically
(Fig. 1C,D). The symmetrical
location of LRC was also observed in dorsal follicles
(Fig. 1E). Thus, in the context
of LRC, `bulge' refers to the permanent portion of the follicle below the
sebaceous glands and not to a physical entity (e.g.
Fig. 1F).
The number of LRC was counted in several anatomic locations within triplets of tail hair follicles (Table 1). While the outer two follicles in each triplet tended to go through the hair cycle concurrently, the central follicle frequently cycled asynchronously and generally had fewer LRC (Fig. 1D). Therefore the central follicles of each triplet were tabulated separately. Occasional LRC were Ki67 positive, indicating that they were capable of dividing (Fig. 1G, inset). The intensity of BrdU labelling varied in different cells, presumably as a result of dilution of the BrdU label during cell division (Fig. 1G). BrdU labelling is therefore described as `heavy' or `light' in Table 1.
In all hair follicles, the highest number of heavily BrdU-labelled cells (range 10-79) was clustered within the noncycling region of the hair follicle beneath the sebaceous glands. This region of the follicle also contained many lightly labelled cells, which indicated that cells in this region were not completely quiescent (Table 1; Fig. 1G). By contrast, nearly all of the BrdU-containing cells located in the cycling region of the hair follicle, extending from the bulge toward the bulb, were lightly labelled and these cells tended to be located individually rather than clustered (Table 1; Fig. 1G). Lightly labelled cells rarely were present above the sebaceous gland (HF infundibulum; Table 1).
Although the greatest number of LRC was located in the non-cycling region of the hair follicle beneath the sebaceous glands, this compartment was not the exclusive location of LRC in tail epidermis. LRC were frequently observed as individual, non-clustered cells within interfollicular epidermis; these cells, like the LRC of the hair follicle, were usually negative for Ki67 (Fig. 1F). LRC occasionally were present in the sebaceous gland (Fig. 1H; Table 1) and, more rarely, in the infundibulum of the hair follicle, near the junction between the follicle and the IFE (Fig. 1G, arrowhead; Table 1). The fact that the LRC were keratin 14 positive was confirmed by double immunolabelling of histological sections of hair follicles (Fig. 1I, arrows). The double-labelled cells were confined to the outer root sheath, indicating that they had not undergone terminal differentiation along any of the hair lineages (Fig. 1I).
Expression of cell lineage markers and putative stem cell
markers
In order to visualise different cellular compartments within the epidermis,
we labelled the wholemounts with a range of antibodies. In contrast to keratin
14, which was uniformly expressed in the basal layer of IFE, the sebaceous
gland and the outer root sheath of hair follicles
(Fig. 2A), keratin 17 labelling
was confined to the HF, extending from below the sebaceous glands to the bulb
(Fig. 2B). Keratin 6 labelling
was restricted to the permanent portion of the hair follicle below the
sebaceous gland, including the bulge region, and thus coincided with the
location of most of the LRC (Fig.
2C). The expression patterns of all three keratins was the same in
tail as in dorsal hair follicles (e.g.
McGowan and Coulombe,
1998).
|
We next examined putative surface markers of epidermal stem cells. ß1
integrins enrich for stem cells in human interfollicular epidermis
(Jones and Watt, 1993;
Jones et al., 1995
;
Jensen et al., 1999
) and the
proteoglycan NG2/MCSP also is upregulated in these cells
(Legg et al., 2003
). In both
mouse and human epidermis, stem cells are included in the population of
keratinocytes that express high levels of the
6ß4 integrin. Mouse
epidermal stem cells are enriched by selection for keratinocytes that have
high levels of
6 integrin expression and low expression of the
transferrin receptor (CD71) (Tani et al.,
2000
). In human epidermis, the combination of high expression of
6 integrin and low expression of a proliferation-associated cell
surface marker (10G7 ag) has been used to isolate epidermal stem cells
(Li et al., 1998
;
Kaur and Li, 2000
).
ß1 integrin expression was uniform in the IFE, sebaceous gland and
most of the ORS, with more intense expression in the bulb of anagen follicles
(Fig. 2G). By contrast,
6 integrin labelling was most intense in the region, including the
bulge, which contained LRC and was otherwise uniform in the IFE and sebaceous
glands (Fig. 2H). An antiserum
to NG2 chondroitin sulphate proteoglycan (NG2; rat homologue of MCSP)
(Nishiyama et al., 1991
),
which crossreacts with the mouse homologue AN2, yielded uniformly weak
labelling in the IFE and sebaceous glands, but there was intense labelling in
the HF extending from below the sebaceous glands to the bulb or just above the
bulb (Fig. 2I). In contrast to
ß1 integrin expression, which was highest in the bulb of anagen
follicles, NG2 expression was higher in the region of the ORS that contained
the LRC.
The marker that showed the best co-localisation with LRC was keratin 15
(Lyle et al., 1998;
Waseem et al., 1999
)
(Fig. 2J, inset; note that the
sebaceous gland labelling is non-specific). Keratin 15 staining was restricted
to the permanent portion of the follicle directly beneath the sebaceous gland
(Fig. 2J) and had a more
restricted distribution than
6ß4
(Fig. 2H) or keratin 6
(Fig. 2C). There was variation
in both the overall intensity of label (most intense during mid to late
anagen) and location (symmetric in some follicles; asymmetric in others)
(Fig. 2J and data not
shown).
In human IFE, there is clustering of the putative stem cells and as a
result the location of the actively cycling and keratin 10-positive cells is
not random, but confined to regions of the basal layer surrounding the stem
cell clusters (Jensen et al.,
1999). The observations that LRC within mouse IFE were scattered
rather than clustered (Fig. 1F)
and that there was no obvious pattern to the location of the Ki67-positive
cells (Fig. 1F) suggested that
the keratin 10-positive basal cells would also be randomly distributed. This
was examined by double labelling wholemounts for keratin 10 and ß1 or
6 integrin (Fig. 2K,L)
and viewing the IFE at high magnification. As predicted, keratin 10-positive
cells were distributed in an apparently random fashion in mouse IFE. As
previously reported for human epidermis, the ß1 integrins were primarily
clustered at the periphery of the ventral plasma membrane of basal cells
(Fig. 2K), while the
6
integrins were localised across the entire membrane surface
(Fig. 2L). The distribution of
6ß4, which is a component of hemidesmosomes, was dense and
somewhat filamentous, in contrast to the punctate distribution of
6ß4 in the ventral membrane of human IFE basal cells
(Jensen et al., 1999
).
Assessing the fate of BrdU-labelled cells during the hair cycle
We were able to exploit the whole-mount labelling method to gain a better
understanding of the fate of LRC during the hair growth cycle. Mice were
sacrificed at the age of 28 days (early anagen), 35 days (anagen) or 42 days
(telogen). Paraffin sections of dorsal and tail skin from the same mice were
examined and the relative percent of follicles in anagen, catagen or telogen
was estimated to confirm the stage of the hair cycle. Although the timing of
entry and exit into the first hair cycle was very tightly regulated in dorsal
epidermis (data not shown), there was more variability in follicles in tail
epidermis. In one study of tail skin, the proportion of follicles ranged from
60-95% anagen at 28 days (n=6 mice), 70-100% anagen and 0-30% telogen
at day 35 (n=3), and 80-90% telogen at day 42 (n=3). Thus,
at each of the selected time points a high percentage of the follicles in the
tail epidermis were at the expected stage of the hair cycle. In addition, the
status of any given follicle was generally obvious from its size and overall
morphology (Fig. 3).
|
We also examined the expression of markers of proliferation and lineage commitment at different phases of the hair growth cycle. During early anagen, a moderate number of cells in the hair matrix at the bulb of the hair follicle expressed the proliferative marker Ki67 (Fig. 3A,B). Ki67 labelling was normally low within the bulge; however, an increase in labelling was observed in anagen follicles and some Ki67-positive LRC were observed (Fig. 3B). Ki67 expression was greatly increased in the bulb during mid to late anagen and there was increased expression throughout the entire cycling region of the ORS (Fig. 3C). By contrast, Ki67 expression was completely absent at the bottom of telogen follicles (Fig. 3D).
The location of apoptotic keratinocytes was monitored by TUNEL labelling.
In early anagen small numbers of scattered TUNEL-positive cells were present,
primarily in the lower third of the follicle
(Fig. 3E). In late anagen
(Fig. 3F, asterisk) and telogen
(Fig. 3G) there were very few
TUNEL-positive cells. In regressing follicles TUNEL-positive cells were
clustered at the bulb (Fig.
3G). These observations are in agreement with studies of the hair
cycle in other body sites (reviewed by
Stenn and Paus, 2001).
During early anagen, CDP was expressed by a few cells at the bottom of the follicles, indicating that differentiation along the IRS and companion layer lineages was being initiated (Fig. 3H). CDP expression in the hair bulb and companion cell layer was markedly increased as anagen progressed (Fig. 3I) and was absent from telogen follicles (Fig. 3J).
In early anagen and telogen follicles 6 integrin expression was
greatest in the region of the follicles containing the LRC
(Fig. 3K,M). As the hair
follicle lengthened during anagen, strong
6 integrin labelling was
maintained in this region but also extended nearly to the bulb
(Fig. 3L); thus, in anagen
follicles,
6 was not a specific marker of the LRC compartment.
Loss of LRC through phorbol ester induced proliferation
The presence of LRC in hair follicles that had undergone repeated cycles of
growth and regression raised the possibility that LRC were incapable of
proliferation. To investigate whether LRC in the tails of 10-month-old mice
(Fig. 4A) could be stimulated
to divide we treated the tails three times per week with the phorbol ester TPA
(Schweizer and Winter, 1982).
By 2 days after initiation of TPA treatment there was increased Ki67 labelling
throughout the IFE, sebaceous glands and along the length of the hair
follicles, indicating widespread stimulation of cell proliferation
(Fig. 4B). By 7 days, there was
a decrease in the number of BrdU-positive cells
(Fig. 4C) and those cells that
did retain label were more lightly labelled
(Fig. 4F) than controls
(Fig. 4G). Individual cells
were positive for BrdU and Ki67 (Fig.
4F, arrowheads). By 12 days, LRC were extremely rare
(Fig. 4D) relative to acetone
treated controls (Fig. 4E). In
contrast to its strong effects on proliferation, TPA did not stimulate
apoptosis, as evaluated by TUNEL labelling
(Fig. 4H-J). These experiments
establish that although LRC can be quiescent for many months, they
nevertheless retain the ability to divide in response to an appropriate
stimulus.
|
K14MycER transgenic mice and nontransgenic littermates were administered
BrdU to mark LRC. After a 70 day chase period, Myc was activated by daily,
topical treatment of tail epidermis with OHT for up to 14 days
(Arnold and Watt, 2001). Mice
were sacrificed at pre-determined time points and wholemounts of tail
epidermis were prepared. Transgenic mice that were not treated with OHT had
normal epidermis (Fig. 5A-C)
(Arnold and Watt, 2001
).
Nontransgenic mice treated for 14 days with OHT also were completely normal
(Fig. 5M-O). Activation of Myc
did not lead to a decrease in the number of LRC in the interfollicular
epidermis or hair follicles by 14 days
(Fig. 5B,E,H,K). By contrast,
expression of Ki67 was increased in the IFE and sebaceous glands as early as
four days after treatment with OHT (Fig.
5F) and was still elevated at 7 and 14 days
(Fig. 5I,L).
|
|
Effects of expressing NLef1 in the epidermis
The second transgenic mouse model we examined is one in which an
N-terminally truncated form of the transcription factor Lef1 is expressed
under the control of the K14 promoter
(Niemann et al., 2002). The
transgene blocks ß-catenin signalling and there is conversion of hair
follicles into cysts of interfollicular epidermis. Mice were examined up to
120 days after labelling (i.e. 132 days after birth). At later stages it was
more difficult to prepare wholemounts because the cysts had become so large
and complex.
Wholemounts revealed that the changes in the hair follicles were much more
extensive and complex than anticipated from analysing Haematoxylin and Eosin
stained skin sections (Fig.
7B,D). Each hair follicle did not give rise to a single cyst, but
to multiple cysts that budded off from the outer root sheath anywhere from
just beneath the sebaceous glands to the bulb of the follicle
(Fig. 7F,H). The first changes
in the mice previously had been observed 6 weeks after birth and were loss of
hair and delayed onset of the first postnatal hair cycle
(Niemann et al., 2002).
However, wholemounts revealed changes in the follicles by 3-4 weeks of age,
consisting of ectopic sebocyte differentiation
(Fig. 7J) and bending of the
base of the follicles associated with early cyst formation
(Fig. 7F, arrows).
|
|
Consistent with the change in differentiation from hair follicle lineages
to IFE, CDP was completely absent from the deformed hair follicles of
transgenic mice (Fig. 7P).
However, those follicles that retained a normal morphology at 3.5 weeks
continued to express CDP (Fig.
7N). In K14NLef1 mice, K10 expression was no longer
restricted to the IFE and upper follicle but was also seen in the lower part
of the deformed hair follicles (arrows in
Fig. 8F). The K10-positive
cells were not in the outer root sheath but were within the inner cell layers
and were often seen in developing cysts
(Fig. 8F, asterisks). In
whole-mount samples from K14
NLef1 mice, keratin 6 was expressed
throughout the structures of the deformed follicles, albeit at low levels
(Fig. 8H). In skin from
transgenic animals, expression of the
6ß4 integrin was no longer
elevated in the upper third of the follicles
(Fig. 8E), but was uniformly
expressed in the hair follicles, sebaceous glands and IFE
(Fig. 8F). No changes in
ß1 integrin expression were observed
(Fig. 8G,H).
Although the increase in number of Ki67-positive cells
(Fig. 8B,D) in K14NLef1
transgenic epidermis relative to controls
(Fig. 8A,C) suggested that LRC
were being lost through proliferation, an alternative possibility was that
they were undergoing apoptosis. In 3.5-week-old transgenics, there was an
increase in TUNEL labelling (Fig.
8I) compared with wild type
(Fig. 8J). However, the
increase was modest and mainly confined to the lower third of the follicle
(Fig. 8I, arrows).
Ki67-positive LRC and cells that were lightly labelled with BrdU were
frequently observed in the transgenic follicles
(Fig. 3K). Thus, although some
LRC may be lost through apoptosis, we believe that in K14
NLef1
transgenics, as in TPA treated wild-type mice
(Fig. 4), most cells lose BrdU
label by undergoing cell division.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Number and location of LRC within the hair follicle
In studies of the hair follicles of mouse back skin, Cotsarelis et al.
(Cotsarelis et al., 1990)
identified LRC as being concentrated in a permanent region of the hair
follicle known as the bulge. As many hair follicles lack a physical bulge in
the outer root sheath, the term `bulge' has come to refer loosely to the
permanent region of the follicle below the sebaceous gland
(Morris and Potten, 1994
;
Narisawa and Kohda, 1996
;
Stenn and Paus, 2001
). In
wholemounts of tail and dorsal follicles, LRC typically were clustered
symmetrically along the entire length of the permanent section of the follicle
below the sebaceous glands. Our observations on the location of hair follicle
LRC are thus in broad agreement with those of others.
The number of LRC per follicle in wholemounts was also in good agreement
with previous estimates. We observed a range of 0 to 15 LRC in dorsal
follicles, consistent with the six to ten reported previously
(Taylor et al., 2000) and the
one to four found in 3D reconstructions of pelage follicles after a prolonged
chase period of 14 months (Morris and
Potten, 1999
). In wholemounts of tail follicles, the number of LRC
was greater (range 10-79 LRC) than in dorsal follicles, which probably
reflects the larger size of the tail follicles. This observation is similar to
the results of Morris and Potten (Morris
and Potten, 1999
), who found 17-45 LRC in the large follicles of
guard hairs.
There are several hypotheses regarding the role that stem cells play during
the hair cycle. According to the bulge activation hypothesis of Cotsarelis et
al. (Cotsarelis et al., 1990),
during late telogen or early anagen, the LRC of the bulge area are activated
to proliferate by signals from the dermal papilla and their progeny migrate to
give rise to the lower follicle. In support of this model, Taylor et al.
(Taylor et al., 2000
) describe
lightly labelled BrdU-positive cells in the lower part of anagen follicles. By
contrast, Oshima et al. (Oshima et al.,
2001
) observed that in vibrissae follicles, which have such a long
anagen that they appear to grow continuously rather than cyclically (J.P.S.,
unpublished), the stem cells themselves migrate to the base of the follicle
before undergoing proliferation. Finally, Panteleyev et al.
(Panteleyev et al., 2001
)
hypothesise that a second reservoir of stem cells, which has migrated from the
bulge during anagen, exists at the periphery of the bulb; these cells survive
catagen and give rise to the hair shaft and inner root sheath cell
lineages.
Our whole-mount data are most consistent with the bulge activation
hypothesis. Although there was normally little proliferative activity in the
zone of LRC in the permanent region of the follicle, some anagen follicles
showed Ki67 labelling in that region and we occasionally observed individual
LRC that were Ki67 positive (e.g. Fig.
3B). Furthermore, cells lightly labelled with BrdU were often
observed in the ORS of the cycling region of anagen follicles and these cells
are most likely to be LRC daughters migrating downwards (e.g.
Fig. 1G). By contrast, heavily
labelled cells rarely were observed in the cycling region of the hair
follicle, suggesting that LRC themselves neither migrate downwards in
appreciable numbers (Oshima et al.,
2001) nor constitute a permanent clustered population within the
bulb (Panteleyev et al.,
2001
). We cannot, of course, rule out the possibility that
unlabelled stem cells migrate to, or are residents of, the bulb.
We saw no obvious decrease in the number of LRC during the course of one
hair growth cycle (Fig. 3)
(Morris and Potten, 1999) and
in mice sacrificed 10 months after labelling LRC were still present, albeit in
smaller numbers and with less intense labelling than in younger animals
(Fig. 4A). These observations
indicate that very few LRC divide during each round of the hair cycle. The
concept that an entire follicle is formed by the progeny of even a single stem
cell would be quite consistent with the known regenerative potential of stem
cells in the haematopoietic system
(Lemischka et al., 1986
).
Equally plausible is the possibility that LRC are only a subset of the stem
cell population within the hair follicle and that the unlabelled stem cells
also contribute daughters to anagen follicles.
The persistence of LRC during successive hair cycles also raised the
possibility that LRC were incapable of division. We were able to rule this out
by applying a strong proliferative stimulus to the epidermis in the form of
repeated applications of TPA (Schweizer
and Winter, 1982). Within 7 days this treatment resulted in a
major reduction in LRC without any corresponding increase in apoptosis. The
loss of LRC correlated with the appearance of lightly labelled BrdU-positive
cells, some of which were Ki67 positive, demonstrating that LRC had been
stimulated to divide.
LRC are present in IFE and sebaceous glands
The existence of LRC within the sebaceous glands and IFE has been
contentious. Cotsarelis et al. (Cotsarelis
et al., 1990) and Taylor et al.
(Taylor et al., 2000
) found no
LRC in the IFE and presented evidence that bulge LRC migrate up the follicle
to populate the sebaceous gland and IFE, implying that there are no stem cells
in the IFE of hair bearing skin. However, this is not consistent with reports
that LRC lie within the IFE and have a patterned distribution corresponding to
the location of epidermal proliferative units, columns of terminally
differentiated cells produced through the proliferation of a central stem cell
surrounded by a ring of transit amplifying cells
(Mackenzie, 1970
;
Christophers, 1971
;
Potten, 1974
;
Morris et al., 1986
).
With whole-mount labelling, it is possible to examine large areas of IFE
and many hair follicles simultaneously. Our observations leave no doubt that
there are scattered LRC in the IFE of tail and dorsal skin. We also found LRC
in the sebaceous glands. There was no evidence that lightly labelled cells
were migrating upwards from the permanent region of the hair follicles,
although such cells were readily detected in the lower part of the follicle.
We did not see any pattern in the distribution of the IFE LRC that would be
suggestive of epidermal proliferative units. In keeping with this, the
K10-positive basal cells also appeared to have a random distribution, in
contrast to their arrangement in human IFE
(Jensen et al., 1999). The
presence of numerous LRC within both tail and dorsal skin IFE suggests that
mouse, like human, IFE is normally maintained by its own stem cell compartment
(Ghazizadeh and Taichman,
2001
; Niemann and Watt,
2002
).
Evaluation of putative stem cell markers
There are two major limitations of label retention as a marker of the stem
cell compartment. First, it cannot be used to isolate viable cell populations;
and, second, it is only a marker of cells that are not dividing. There is
therefore a pressing need for stem cell-specific surface markers. Wholemounts
provide a rapid screen for candidate markers.
The best surface marker of those evaluated was 6ß4 integrin,
which was specifically upregulated in the LRC zone of early anagen and telogen
follicles. Keratin 15 (Lyle et al.,
1998
) showed even better colocalisation with LRC, yet neither
6ß4 nor keratin 15 was exclusively upregulated on individual LRC.
More work is required to determine whether or not the keratin 15-positive,
high
6ß4-expressing keratinocytes in the bulge that are not LRC
are also stem cells. One aspect of
6ß4 expression that had not
been observed previously is that the zone of high expression extends to the
bulb during anagen. This demonstrates that expression of putative stem cell
markers can be highly dynamic and it is intriguing to speculate that the
change in
6ß4 expression reflects movement of committed progenitor
cells downwards from the LRC zone.
Manipulation of the LRC compartment and lineage commitment in
transgenic mice
We used the whole-mount method to visualise the changes induced in the
epidermis by activating Myc or expressing NLef1. In cultured human
keratinocytes, activation of Myc results in depletion of the stem cell
compartment, as evaluated by an increase in abortive clones of terminally
differentiated cells (Gandarillas and
Watt, 1997
). In transgenic mice in which Myc is constitutively
expressed in the epidermal basal layer, there is a depletion of LRC in the IFE
(Waikel et al., 2001
).
However, after 14 days of Myc activation in the tails of adult mice, we did
not see any loss of LRC, demonstrating that LRC are not required to drive the
marked hyperproliferation of IFE that is a feature of this transgenic model
(Arnold and Watt, 2001
).
Instead, recruitment of LRC into cycle must occur after the initial wave of
proliferation induced by Myc (Waikel et
al., 2001
).
What was most striking in the wholemounts of K14MycER epidermis was the
increased sebocyte differentiation and disorganisation, which were far more
extensive than appreciated from histological sections
(Arnold and Watt, 2001). Nile
Red staining revealed that the increased sebaceous differentiation was not
confined to the hair follicles, but also occurred in the IFE. This suggests
that Myc not only stimulates sebocyte differentiation at the expense of the
hair lineages, but may also reprogram IFE keratinocytes to differentiate along
the sebocyte lineage. In cultured human keratinocytes, Myc activation is not
sufficient to induce conversion to sebocytes
(Gandarillas and Watt, 1997
).
However, activation of Myc in keratinocytes in vivo causes changes in gene
expression in the underlying dermis (Frye
et al., 2003
), which may facilitate reprogramming. What is clear
is that Myc induced sebaceous differentiation occurs independently of LRC
proliferation or terminal differentiation.
Expression of NLef1 blocks ß-catenin signalling and thereby
converts hair follicles into cysts of IFE
(Huelsken et al., 2001
;
Merrill et al., 2001
;
DasGupta et al., 2002
;
Niemann et al., 2002
).
Consistent with the change in differentiation programme from hair lineages to
IFE (Niemann et al., 2002
),
CDP was absent from the aberrant follicles and keratin 10 labelling was
observed in the cysts of K14
NLef1 mice. Wholemounts revealed that
changes in the
NLef1 transgenics took place much earlier and were more
extensive than we had thought previously. By 3.5 weeks after birth, there were
numerous cysts developing along the length of the ORS, demonstrating that
reprogramming was neither confined to the LRC zone nor to cells in contact
with the dermal papilla.
In K14NLef1 transgenics, as in the K14MycER mice, IFE
hyperproliferation occurred without a corresponding loss of LRC. However, LRC
were almost completely absent from the hair follicles and IFE in 3.5-month-old
mice. Although a small increase in apoptosis was observed in K14
NLef1
mice compared with nontransgenic mice, the apoptotic cells were mainly located
in the lower follicle. By contrast, Ki67-positive LRC were present in the
upper follicle, suggesting that most of the LRC are likely to be lost through
proliferation. In spite of the loss of LRC there was no evidence of stem cell
depletion as the IFE remained healthy and differentiated normally, even in
mice older than 2 years (Niemann et al.,
2002
).
In conclusion, the whole-mount labelling method has allowed us to clarify a
number of issues in epidermal stem cell biology. It will be of great use in
the search for stem cell markers and in characterising the skin of genetically
modified mice. Our data support the bulge activation hypothesis of cyclical
hair growth, but demonstrate that there are distinct LRC populations in the
IFE and sebaceous glands. LRC can readily be stimulated to divide in response
to TPA, yet Myc induced hyperproliferation and sebocyte differentiation can
occur without changes to the LRC compartment. The entire ORS is responsive to
NLef1 induced IFE and sebocyte differentiation and loss of LRC does not
compromise epidermal integrity. Finally, our results show that LRC are not
synonymous with epidermal stem cells, but rather represent a subset of the
stem cell population.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akiyama, M., Smith, L. T. and Shimizu, H.
(2000). Changing patterns of localization of putative stem cells
in developing human hair follicles. J. Invest.
Dermatol. 114,321
-327.
Arnold, I. and Watt, F. M. (2001). c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny. Curr. Biol. 11,558 -568.[CrossRef][Medline]
Bannigan, J. G., Cottell, D. C. and Morris, A. (1990). Study of the mechanisms of BUdR-induced cleft palate in the mouse. Teratology 42, 79-89.[Medline]
Barrandon, Y. and Green, H. (1987). Three clonal types of keratinocyte with different capacities for multiplication. Proc. Natl. Acad. Sci. USA 84,2302 -2306.[Abstract]
Bickenbach, J. R. (1981). Identification and behavior of label-retaining cells in oral mucosa and skin. J. Dent. Res. 60C,1611 -1620.[Medline]
Bickenbach, J. R. and Chism, E. (1998). Selection and extended growth of murine epidermal stem cells in culture. Exp. Cell Res. 244,184 -195.[CrossRef][Medline]
Bickenbach, J. R., McCutecheon, J. and Mackenzie, I. C. (1986). Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissues. Cell Tiss. Kinet. 19,325 -333.[Medline]
Catalano, R. A. and Lillie, R. D. (1975). Elimination of precipitates in Oil Red O fat stain by adding dextrin. Stain Techol. 50,297 -299.
Christophers, E. (1971). The columnar structure of the epidermis. Possible mechanism of differentiation. Z. Zellforsch. Mikrosk. Anat. 114,441 -450.[Medline]
Cotsarelis, G., Sun, T. T. and Lavker, R. M. (1990). Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61,1329 -1337.[Medline]
DasGupta, R., Rhee, H. and Fuchs, E. (2002). A
developmental conundrum: a stabilized form of beta-catenin lacking the
transcriptional activation domain triggers features of hair cell fate in
epidermal cells and epidermal cell fate in hair follicle cells. J.
Cell Biol. 158,331
-344.
Ellis, T., Gambardella, L., Horcher, M., Tschanz, S., Capol, J.,
Bertram, P., Jochum, W., Barrandon, Y. and Busslinger, M.
(2001). The transcriptional repressor CDP (Cutl1) is essential
for epithelial cell differentiation of the lung and the hair follicle.
Genes Dev. 15,2307
-2319.
Ferraris, C., Bernard, B. A. and Dhouailly, D. (1997). Adult epidermal keratinocytes are endowed with pilosebaceous forming abilities. Int. J. Dev. Biol. 41,491 -498.[Medline]
Frye, M., Gardner, C., Li, E. R., Arnold, I., and Watt, F.
M. (2003). Evidence that Myc activation depletes the
epidermal stem cell compartment by modulating adhesive interactions with the
local microenvironment. Development
130,2793
-2808.
Fuchs, E., Merrill, B. J., Jamora, C. and DasGupta, R. (2001). At the roots of a never-ending cycle. Dev. Cell 1,13 -25.[Medline]
Fuchs, E. and Raghavan, S. (2002). Getting under the skin of epidermal morphogenesis. Nat. Rev. Genet. 3,199 -209.[CrossRef][Medline]
Gandarillas, A. and Watt, F. M. (1997). c-Myc
promotes differentiation of human epidermal stem cells. Genes
Dev. 11,2869
-2882.
Ghazizadeh, S. and Taichman, L. B. (2001).
Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of
adult mouse skin. EMBO J.
20,1215
-1222.
Hamilton, E. and Potten, C. S. (1972). Influence of hair plucking on the turnover time of the epidermal basal layer. Cell Tiss. Kinet. 5,505 -517.[Medline]
Hardy, M. H. (1992). The secret life of the hair follicle. Trends Genet. 8, 55-61.[Medline]
Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G. and Birchmeier, W. (2001). beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 105,533 -545.[CrossRef][Medline]
Jensen, U. B., Lowell, S. and Watt, F. M.
(1999). The spatial relationship between stem cells and their
progeny in the basal layer of human epidermis: a new view ased on whole-mount
labelling and lineage analysis. Development
126,2409
-2418.
Jones, P. H. and Watt, F. M. (1993). Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73,713 -724.[Medline]
Jones, P. H., Harper, S. and Watt, F. M. (1995). Stem cell patterning and fate in human epidermis. Cell 80,83 -93.[Medline]
Kaur, P. and Li, A. (2000). Adhesive properties
of human basal epidermal cells: an analysis of keratinocyte stem cells,
transit amplifying cells, and postmitotic differentiating cells. J.
Invest. Dermatol. 114,413
-420.
Lavker, R. M., Miller, S., Wilson, C., Cotsarelis, G., Wei, Z. G., Yang, J. S. and Sun, T. T. (1993). Hair follicle stem cells: their location, role in hair cycle, and involvement in skin tumor formation. J. Invest. Dermatol. 101,16S -26S.[Medline]
Legg, J., Jensen, U. B., Broad, S., Leigh, I. and Watt, F. M. (2003). Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis. Development (in press).
Leigh, I. M., Purkis, P. E., Whitehead, P. and Lane, E. B. (1993). Monospecific monoclonal antibodies to keratin 1 carboxy terminal (synthetic peptide) and to keratin 10 as markers of epidermal differentiation. Br. J. Dermatol. 129,110 -119.[Medline]
Lemischka, I. R., Raulet, D. H. and Mulligan, R. C. (1986). Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 45,917 -927.[Medline]
Li, A., Simmons, P. J. and Kaur, P. (1998).
Identification and isolation of candidate human keratinocyte stem cells based
on cell surface phenotype. Proc. Natl. Acad. Sci. USA
95,3902
-3907.
Lyle, S., Christofidou-Solomidou, M., Liu, Y., Elder, D. E.,
Albelda, S. and Cotsarelis, G. (1998). The C8/144B
monoclonal antibody recognizes cytokeratin 15 and defines the location of
human hair follicle stem cells. J. Cell Sci.
111,3179
-3188.
Mackenzie, I. C. (1970). Relationship between mitosis and the ordered structure of the stratum corneum in mouse epidermis. Nature 226,653 -655.[Medline]
McGowan, K. M. and Coulombe, P. A. (1998).
Onset of keratin 17 expression coincides with the definition of major
epithelial lineages during skin development. J. Cell
Biol. 143,469
-486.
Merrill, B. J., Gat, U., DasGupta, R. and Fuchs, E.
(2001). Tcf3 and Lef1 regulate lineage differentiation of
multipotent stem cells in skin. Genes Dev.
15,1688
-1705.
Morris, R. J., Fischer, S. M. and Slaga, T. J. (1985). Evidence that the centrally and peripherally located cells in the murine epidermal proliferative unit are two distinct cell populations. J. Invest. Dermatol. 84,277 -281.[Abstract]
Morris, R. J., Fischer, S. M. and Slaga, T. J. (1986). Evidence that a slowly cycling subpopulation of adult murine epidermal cells retains carcinogen. Cancer Res. 46,3061 -3066.[Abstract]
Morris, R. J. and Potten, C. S. (1994). Slowly cycling (label-retaining) epidermal cells behave like clonogenic stem cells in vitro. Cell Prolif. 27,279 -289.[Medline]
Morris, R. J. and Potten, C. S. (1999). Highly
persistent label-retaining cells in the hair follicles of mice and their fate
following induction of anagen. J. Invest. Dermatol.
112,470
-475.
Narisawa, Y. and Kohda, H. (1996). Two- and three-dimensional demonstrations of morphological alterations of early anagen hair follicle with special reference to the bulge area. Arch. Dermatol. Res. 288,98 -102.[CrossRef][Medline]
Niemann, C. and Watt, F. M. (2002). Designer skin: lineage commitment in postnatal epidermis. Trends Cell Biol. 12,185 -192.[CrossRef][Medline]
Niemann, C., Owens, D. M., Hulsken, J., Birchmeier, W. and Watt,
F. M. (2002). Expression of DeltaNLef1 in mouse epidermis
results in differentiation of hair follicles into squamous epidermal cysts and
formation of skin tumours. Development
129,95
-109.
Nishiyama, A., Dahlin, K. J., Prince, J. T., Johnstone, S. R. and Stallcup, W. B. (1991). The primary structure of NG2, a novel membrane-spanning proteoglycan. J. Cell Biol. 114,359 -371.[Abstract]
Oshima, H., Rochat, A., Kedzia, C., Kobayashi, K. and Barrandon, Y. (2001). Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104,233 -245.[CrossRef][Medline]
Panteleyev, A. A., Jahoda, C. A. and Christiano, A. M.
(2001). Hair follicle predetermination. J. Cell
Sci. 114,3419
-3431.
Potten, C. S. (1974). The epidermal proliferative unit: the possible role of the central basal cell. Cell Tiss. Kinet. 7,77 -88.[Medline]
Potten, C. S. and Morris, R. J. (1988). Epithelial stem cells in vivo. J. Cell Sci. Suppl. 10, 45-62.[Medline]
Regnier, M., Vaigot, P., Darmon, M. and Prunieras, M. (1986). Onset of epidermal differentiation in rapidly proliferating basal keratinocytes. J. Invest. Dermatol. 87,472 -476.[Abstract]
Schweizer, J. and Marks, F. (1977a). A developmental study of the distribution and frequency of Langerhans cells in relation to formation of patterning in mouse tail epidermis. J. Invest. Dermatol. 69,198 -204.[Medline]
Schweizer, J. and Marks, F. (1977b). Induction of the formation of new hair follicles in mouse tail epidermis by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 37,4195 -4201.[Abstract]
Schweizer, J. and Winter, H. (1982). Changes in regional keratin polypeptide patterns during phorbol ester-mediated reversible and permanently sustained hyperplasia of mouse epidermis. Cancer Res. 42,1517 -1529.[Medline]
Schweizer, J., Kinjo, M., Furstenberger, G. and Winter, H. (1984). Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis: basal cells with properties of terminally differentiating cells. Cell 37,159 -170.[Medline]
Shah, R. M. and MacKay, R. A. (1978). Teratological evaluation of 5-fluorouracil and 5-bromo-2-deoxyuridine on hamster fetuses. J. Embryol. Exp. Morphol. 43, 47-54.[Medline]
Stenn, K. S. and Paus, R. (2001). Controls of
hair follicle cycling. Physiol. Rev.
81,449
-494.
Sundberg, J. P., Boggess, D., Sundberg, B. A., Beamer, W. G. and Shultz, L. D. (1993). Epidermal dendritic cell populations in the flaky skin mutant mouse. Immunol. Invest. 22,389 -401.[Medline]
Sundberg, J. P., Hogan, M. E. and King, L. E. (1996). Normal biology and aging changes in skin and hair. In Pathobiology of the Aging Mouse (ed. U. Mohr, D. L. Dungworth, C. C. Capen, W. W. Carlton, J. P. Sundberg and J. M. Ward), pp.303 -323. Washington DC: ILSI Press.
Sundberg, J. P., Rourk, M. H., Boggess, D., Hogan, M. E., Sundberg, B. A. and Bertolino, A. P. (1997). Angora mouse mutation: altered hair cycle, follicular dystrophy, phenotypic maintenance of skin grafts, and changes in keratin expression. Vet. Pathol. 34,171 -179.[Abstract]
Sundberg, J. P. and King, L. E. (2000). Skin and its appendages: normal anatomy and pathology of spontaneous transgenic and targeted mouse mutations. In Pathology of Genetically Engineered Mice (ed. J. M. Ward, J. F. Mahler, R. R. Maronpot and J. P. Sundberg), pp. 181-213. Ames: Iowa State University Press.
Tani, H., Morris, R. J. and Kaur, P. (2000).
Enrichment for murine keratinocyte stem cells based on cell surface phenotype.
Proc. Natl. Acad. Sci. USA
97,10960
-10965.
Taylor, G., Lehrer, M. S., Jensen, P. J., Sun, T. T. and Lavker, R. M. (2000). Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102,451 -461.[Medline]
Von Ballestrem, C. G., Uniyal, S., McCormick, J. I., Chau, T., Singh, B. and Chan, B. M. (1996). VLA-beta 1 integrin subunit-specific monoclonal antibodies B1.1 and MB1.2: binding to epitopes not dependent on thymocyte development or regulated by phorbol ester and divalent cations. Hybridoma 15,125 -132.[Medline]
Waikel, R. L., Kawachi, Y., Waikel, P. A., Wang, X. J. and Roop, D. R. (2001). Deregulated expression of c-Myc depletes epidermal stem cells. Nat. Genet. 28,165 -168.[CrossRef][Medline]
Waseem, A., Dogan, B., Tidman, N., Alam, Y., Purkis, P.,
Jackson, S., Lalli, A., Machesney, M. and Leigh, I. M.
(1999). Keratin 15 expression in stratified epithelia:
downregulation in activated keratinocytes. J. Invest.
Dermatol. 112,362
-369.
Watt, F. M. (1998). Epidermal stem cells: markers, patterning and the control of stem cell fate. Philos. Trans. R. Soc. Lond. B Biol. Sci.353,831 -837.[CrossRef][Medline]
Zhang, W., Remenyik, E., Zelterman, D., Brash, D. E. and
Wikonkal, N. M. (2001). Escaping the stem cell
compartment: sustained UVB exposure allows p53- mutant keratinocytes to
colonize adjacent epidermal proliferating units without incurring additional
mutations. Proc. Natl. Acad. Sci. USA
98,13948
-13953.