1 Institute for Molecular Bioscience, and Special Research Centre for Functional
and Applied Genomics, University of Queensland, and the Cooperative Research
Centre for the Discovery of Genes for Common Human Diseases, Victoria,
Australia
2 Epithelial Stem Cell Biology Laboratory, Peter MacCallum Cancer Institute,
Melbourne, Victoria, Australia
Author for correspondence (e-mail:
B.Wainwright{at}imb.uq.edu.au)
Accepted 22 July 2004
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SUMMARY |
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Key words: Shh, Epidermis, Stem cells, BCC, Proliferation, Dhh, Ihh
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Introduction |
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In order to determine the tumorigenic potential of Shh, Dhh and Ihh, and to establish whether they play a role in regulating the epidermal stem cell population of the interfollicular, hair follicle or sebaceous lineages, we overexpressed each of the hedgehog genes in the basal layer of the skin via the keratin 14 promoter. A subset of Shh and Dhh transgenic embryos presented with a marked expansion of epidermal progenitor cells, an increase in basal cell proliferative activity and a delay in basal cell differentiation. In addition, we observed several Shh and Dhh transgenic embryos that lacked epidermal proliferative activity and cell/tissue renewal potential. Our results show for the first time that hedgehog signalling in the proliferative compartment of the epidermis plays an important role in regulating homeostatic cell renewal of the skin, and indicate that manipulation of the hedgehog pathway may provide a useful tool for the experimental investigation of epidermal tissue regeneration and for the ex-vivo expansion of epidermal progenitors for clinical applications.
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Materials and methods |
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Histology and immunohistochemistry
Back skin from E17.5 or E18.5 embryos were fixed overnight in 4%
paraformaldehyde in PBS, and embedded in paraffin. Histological analysis was
performed on 4 µm sections stained with haematoxylin and eosin. Antibody
markers were analysed on 4 µm tissue sections via standard
immunofluorescence and immunohistochemistry techniques using the following
antibodies: K14 (1/10,000), K6 (1/500), K10 (1/500) loricrin (1/500) (Babco),
Ki67 (1/500) (Novo Castra), PCNA (1/100) (ZYMED laboratories), p63 (1/100)
(gift from Dr F. McKeon), Ihh (1/30) (C-15; Santa Cruz).
In situ hybridisation
RNA in situ analyses were performed on 14 µm paraffin-embedded skin
sections using Digoxigenin (DIG)-labelled RNA probes. Sections were rinsed in
xylene then hydrated through a graded ethanol series. Sections were treated
with 10 µg/ml proteinase K for 10 minutes at 37°C, rinsed in PBS, fixed
in 4% PFA for 10 minutes, rinsed in PBS, acetylated in 0.1 M
triethanolamine/0.25% acetic anhydride for 10 minutes. Sections were then
dehydrated through a graded ethanol series and xylene, then allowed to air
dry. Then 500 µg of RNA probe plus 1 mg/ml of salmon sperm were heated at
80°C for 5 minutes, and added to 500 µl of hybridisation solution
solution (50% formamide, 10% dextran sulphate, 10 mM Tris-HCl, 10 mM EDTA, 0.6
M NaCl, 10 mM dithiothreitol (DTT), 1xDenhardt's solution, 0.25% sodium
dodecyl-sulphate (SDS) and 200 µg/ml yeast total RNA). RNA probes were
hybridised overnight at 64°C in a humidified chamber. Washing and blocking
steps were performed using DIG wash and block buffer set (Roche) as per
manufacturer's instructions. Colour detection was performed using Nitro-blue
tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) in the
presence of 50 mM MgCl2 and 10% polyvinyl alcohol. Samples were
then fixed in 8% formamide for 60 minutes, dehydrated through a graded ethanol
series and xylene, and mounted in xylene-based mounting media. Transgene RNA
expression was detected using a 328 bp antisense RNA probe, made from PCR
amplification of the K14 poly A region of the promoter (see primer sequences
above). Ptc2, Gli1, Gli2, Gli3 and Shh RNA probes were
kindly provided by Dr C. C. Hui. The Ptc1 probe has been previously
described (Hahn et al.,
1996).
Skin grafts
Skin samples for grafting were prepared and grafted onto 6- to 10-week-old
female, SCID recipient mice as previously described
(Mayumi et al., 1988).
Recipient mice were shaved, and the graft dressed and bandaged. Dressings were
removed seven days post-graft and skin phenotype noted weekly.
Keratinocyte cell culture
Primary keratinocytes were isolated from the skin of E18.5 embryos as
described by Hager et al. (Hager et al.,
1999). Proliferative basal cells were selected via rapid
attachment (10 minutes) onto vitrogen/fibronectin-coated 60 mm dishes and
cultured in keratinocyte growth media (EMEM, 8% chelex-treated fetal bovine
serum, 4 ng/ml epidermal growth factor (EGF) and 50% fibroblast conditioned
media), with media changes every second day. After 4 weeks of incubation,
cells were fixed in 100% ethanol at 20°C for 20 minutes, and
stained with 1% crystal violet.
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Results |
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Shh pathway activity regulates the fate of epidermal progenitor cells
In order to further characterise the proliferative potential of
ShhWrinkled and ShhTranslucent
epidermis, we analysed the expression of the transcription factor p63 which is
normally expressed in a subset of embryonic epidermal basal cells
(Fig. 5A), and is thought to
maintain the proliferative potential of basal cells
(Koster and Roop, 2004;
McKeon, 2004
). p63 expression
can therefore be used to mark epidermal progenitor cells. We observed a
substantial increase in the number of basal cells expressing p63 in
ShhWrinkled epidermis, with expanded p63 expression
detected in up to four basal cell layers
(Fig. 5C). In order to
determine whether Shh activity augments epidermal proliferative activity by
directly increasing the pool of epidermal progenitor cells, we analysed the
clonogenic growth potential of ShhWrinkled keratinocytes
using a well-characterised in vitro cell culture assay system
(Bickenbach and Chism, 1998
;
Kaur and Li, 2000
). Primary
keratinocytes from E18.5 ShhWrinkled transgenic embryos
and wild-type littermate controls were plated at 106 per 60 mm dish
and the epidermal progenitor population enriched for by rapid attachment (10
minutes) to collagenI/fibronectin-coated dishes. Cell cultures were analysed
following 4 weeks of cultivation. The results from three independent
experiments revealed that ShhWrinkled keratinocytes
consistently gave rise to greater numbers of colonies compared to wild-type
controls, i.e. 151±19 versus 41±4, respectively, reflecting a
three-fold increase in colony numbers from ShhWrinkled
skin. In addition, ShhWrinkled keratinocyte cultures
displayed an increase in colony density with respect to wild-type controls
(represented in Fig. 6C and
Fig. 6B, respectively), thereby
supporting our observation that ShhWrinkled keratinocytes
are hyperproliferative. These data therefore show that Shh can induce
epidermal progenitor cell hyperplasia and that Shh promotes the proliferation
of epidermal stem cells and their immediate progeny. In contrast,
ShhTranslucent embryos exhibited a complete absence of
epidermal p63 expression (Fig.
5D). We therefore performed in vitro keratinocyte analyses in
order to determine the growth potential of ShhTranslucent
epidermis. Consistent with the observation that
ShhTranslucent skin exhibits a severely depleted basal
compartment we obtained a limited (1x103) number of primary
keratinocytes, which failed to attach, survive or grow, under the same
conditions used to culture wild-type and ShhWrinkled
keratinocytes. The inability of ShhTranslucent embryonic
back skin to graft onto SCID mice (0/14) also supports a lack of epidermal
regeneration capacity. We also speculate that epidermal stem cells of the
bulge are absent in ShhTranslucent skin, given the absence
of any hair follicle structures in these mice. Definitive evidence of the
absence of follicular stem cells requires the identification of appropriate
markers for these cells. Although CD34 has recently been shown to be a marker
for the adult murine hair follicle bulge region
(Trempus et al., 2003
), and
although we could detect the expression of CD34 in wild-type adult epidermis,
CD34 was not expressed in embryonic (E18.5) epidermis (data not shown),
suggesting that it is not a suitable marker for the embryonic follicular stem
cell compartment.
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The level of ectopic Shh activity directs epithelial cell response
The observation that hK14-Shh transgenics present with a range of
skin and developmental phenotypes that appeared to increase in severity from
ShhRegular, to ShhWrinkled, to
ShhTaut and finally ShhTranslucent,
suggests that each phenotype may result from increasing levels of hedgehog
transgene activity. RNA in situ analysis showed an increase in transgene
expression from ShhRegular, through
ShhWrinkled, to ShhTaut
(Fig. 7A-C), as determined by a
transgene-specific RNA probe. However, transgene analysis was hindered in the
ShhTranslucent phenotype due to the severe depletion of
basal cells in which transgene expression is directed
(Fig. 7D). Genomic PCR analyses
were used in order to determine the relative copy number associated with each
skin phenotype. PCR analyses indicate an increase in transgene copy number
from ShhRegular, to ShhWrinkled, to
ShhTaut, thereby supporting our RNA in situ expression
level data. In addition, genomic copy number analyses indicate that
ShhTranslucent embryos have a consistently higher
transgene copy number than other phenotypes
(Fig. 7E).
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Twenty-eight independent Ihh transgenic embryos were produced and
no overt HF, sebaceous or interfollicular epidermal phenotypes were observed
(hence 28 IhhRegular transgenic). Although Ihh
overexpression did not result in an epidermal phenotype, it gave rise to a
mild embryonic limb phenotype (refer to
Fig. 1B-D). Previously
published data have shown that although both Shh and Dhh have strong
polarising activity in the developing limb bud, Ihh (even at high
concentrations) gives rise to only minor digit duplications of the chick wing
(Pathi et al., 2001). These
results support our observations that Ihh, although capable of inducing the
same phenotype as low levels of Shh activity, is not homologous with the
ability of Shh to induce strong polarising effects in the limb. In support of
Ihh functional activity we observed high levels of Ihh transgene
(Fig. 8A) and Ihh protein
(Fig. 8B) expression within the
basal layer of IhhRegular transgenic epidermis. In
addition, we observed high levels of endogenous Shh
(Fig. 8C) and Ptc1
(Fig. 8D) expression in the
basal layer and high levels of Gli1 and Gli2 expression in the HFs of
IhhRegular skin. Since Ihh overexpression had no effect on
embryonic epidermal development, we grafted E18.5 back skin from 18
IhhRegular transgenic embryos onto SCID recipient mice, in
order to determine whether ectopic Ihh activity had any effect on adult
epidermal homeostasis. Grafts were monitored weekly for 6 months and both
macroscopic and histological analysis revealed epidermal morphology
indistinguishable from wild-type graft controls (data not shown). We have
therefore shown that Ihh pathway activity does not induce an epidermal
phenotype and it is therefore not functionally redundant with Shh. In
addition, our data suggest that epidermal responsiveness to the hedgehog
signal lies downstream of Gli gene transcriptional regulation.
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Discussion |
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Given the perinatal lethality associated with our embryonic phenotypes, the
production of viable conditional Shh overexpression mouse model systems is
required in order to analyse the progression/regression of the stem/progenitor
cell population over time. An inducible conditional Shh overexpression system
would also allow the documentation of precise timing of transgene expression
onset, thus elucidating the role of heterochronic transgene expression in
producing a given skin phenotype. We are currently in the process of
transgenic animal production, which will allow us to further analyse the role
of Shh and Dhh target genes in regulating epidermal basal
cell homeostasis. However, the data presented here show a consistent trend
between the level of Shh activity and the resulting skin phenotype, suggesting
that the difference between the observed phenotypes is probably related to the
level of transgene expression, with the Translucent phenotype
resulting from the highest level of Shh expression. Such a mechanism would be
consistent with the known dose dependency of Shh action in tissues such as the
developing central nervous system. This is also consistent with the
observation that different levels of hedgehog signalling activity give rise to
different skin phenotypes and skin lesions
(Grachtchouk et al., 2003). The
loss of epidermal progenitor cells in ShhTranslucent
epidermis may be due to the exhaustion of the stem cell compartment; however,
given that almost no basal cells remain, it is possible that high levels of
ectopic Shh activity act to inhibit epidermal stem cell fate.
Hedgehog-induced proliferation is restricted to the epidermal basal cell compartment: the cellular origin of hedgehog-induced skin tumours
Previous overexpression studies have shown that elevated hedgehog pathway
activity in the basal cells of the skin, using basal specific promoters (K5
and K14), gives rise to epidermal anomalies and BCC-like lesion formation
(Grachtchouk et al., 2003;
Oro et al., 1997
;
Sheng et al., 2002
;
Xie et al., 1998
). However, we
have recently shown that overexpression of hedgehog in only a subset of basal
cells, using the human K1 promoter (hK1), results in the ablation of embryonic
hair follicle development and does not give rise to BCC
(Ellis et al., 2003
). The
overexpression of hedgehog via the hK1 promoter drives hedgehog activity in
committed epidermal cells, thus only in a small subset (20-30%) of basal
cells, the PMD-population. In contrast, the overexpression of hedgehog via the
hK14 promoter drives Shh activity in all basal cell populations. The
hedgehog-induced skin phenotype differences observed in hK14-Shh and
hK1-Hh transgenic mice could be attributed to the timing at which
hedgehog activity is induced by the keratin promoters (hK14: E9.5 versus hK1:
E12.5). However, in view of the data presented here, it is a possibility that
the major contributing factor is the activity of the hedgehog pathway in the
epidermal progenitor compartment.
Although BCCs are the most common form of human skin cancer, much remains
to be elucidated about the aetiology, including the cellular origin. The outer
root sheath (ORS) cells of the hair follicles are strong candidates given that
BCCs are seldom observed in hairless regions of the body and appear to
invaginate from structures resembling HFs
(Weedon, 1981). BCC also has
an immature, stem cell-like appearance leading to the suggestion that BCC
originates from aberrant Shh activity within the stem cell population of the
interfollicular epidermis or those of the hair follicle bulge
(Taipale and Beachy, 2001
;
Zhang and Kalderon, 2001
). We
observed hK14-Shh and hK14-Dhh embryonic skin either
enriched or devoid of epidermal progenitor cell activity and/or hair follicles
is equally competent to produce BCC. We hypothesise that hedgehog induces BCC
transformation via promoting the proliferation and subsequent invagination of
cells residing in the basal compartment. This is consistent with the
proliferative effect of Shh on epidermal cells
(Fan and Khavari, 1999
;
Fan et al., 1997
;
Morgan et al., 1998
;
Oro et al., 1997
;
Sato et al., 1999
), and the
role it plays during invagination of the developing hair follicle
(Chiang et al., 1999
;
St-Jacques et al., 1998
).
Although we can not rule out the possibility that stem cells sustain genetic
mutations that predispose their progeny to transformation, the results
presented here suggest that neither stem cells nor ORS cells of the hair
follicle are required for BCC formation. Although both stem cells and ORS
cells reside in the basal compartment, and, according to our hypothesis can
act as the cellular origin to hedgehog pathway-induced skin tumours, our data
show that BCC transformation can occur in basal cells lacking inherent
self-renewing potential. Consistent with this suggestion, only 26% of human
BCCs exhibit p63 expression (Dellavalle et
al., 2002
), thus representing the subset of BCCs that arose within
the epidermal progenitor compartment.
Dhh, but not Ihh, is a functional homologue of Shh signalling in the skin
In vitro biochemical evidence has shown that Shh, Dhh and Ihh have equal
binding affinities for the Ptc1 receptor thereby suggesting that redundancy
between hedgehog protein activity may exist
(Carpenter et al., 1998). In
vitro data have since identified a degree of functional homology amongst Shh
and Ihh proteins in hypertrophic chondrocyte differentiation
(Vortkamp et al., 1996
) and
osteoblastic differentiation (Kinto et
al., 1997
), and among Shh, Dhh and Ihh proteins during motor
neuron induction and polarisation of the developing limb
(Pathi et al., 2001
). In order
to further characterise the role of Shh signalling in maintaining the
epidermal stem cell population and promoting basal cell proliferation, we
overexpressed the mammalian homologues Dhh and Ihh in the basal cells of mouse
skin and observed that Dhh, but not Ihh, is a functional homologue of Shh
activity during epidermal stem cell homeostasis and skin tumour formation.
Recent work by Watt and colleagues
(Niemann et al., 2003
) led to
the hypothesis that Ihh plays a role in the proliferation of sebaceous cells,
given the expression of Ihh in sebaceous tumours of
K14-
N-Lef1 mice. Our K14-Ihh transgenic
epidermis directly tests this hypothesis, and despite ectopic expression of
Ihh protein in the basal cells of IhhRegular epidermis and
activation of Shh and Ptc1 expression, there was no evidence of hair follicle
or sebaceous lineage defects, nor any overt proliferative activity. These data
suggest that the expression of Ihh within K14-
N-Lef1
mice is not causative of sebaceous hyperplasia, but rather a consequence of
inappropriate ß-catenin signalling.
Our observation that Dhh overexpression gives rise to embryo phenotypes indistinguishable from Shh overexpression shows that Dhh is a functional homologue of Shh in epithelial cells, and in particular, that ectopic Dhh pathway activation plays a role in regulating the proliferation of the epidermal basal cell compartment. Given that cancer can be seen as a disease of unregulated cell renewal, an understanding of the mechanisms underlying epidermal proliferative activity is fundamental to understanding how cancer cells obtain unlimited proliferative potential. The data presented here show that both Shh and Dhh signalling activity can result in epidermal hyperplasia or hypoplasia. Thus the elucidation of those targets common to both Shh and Dhh signalling may help identify the genes involved in epidermal cell renewal and those genes responsible for BCC formation. Our data also suggest that the current deficiencies in effective epidermal cell culture for tissue therapies might usefully be addressed by manipulation of the hedgehog pathway, such that the magnitude and timing of Shh activity can be used to drive basal cell proliferation, resulting in increased epidermal proliferation without disrupting epidermal stratification.
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ACKNOWLEDGMENTS |
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Footnotes |
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