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First published online 4 July 2007
doi: 10.1242/dev.02868
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Department of Molecular Biology and Pharmacology, and Division of Dermatology, Department of Medicine, Washington University School of Medicine, Box 8103, 660 South Euclid Avenue, Saint Louis, MO 63110, USA.
Author for correspondence (e-mail:
kopan{at}wustl.edu)
Accepted 16 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hair follicle, Notch1, Keratinocyte, Kitl (Scf), Tgfß, IGFBP, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Millar,
2002; Sengel,
1976). Migratory melanocytes, and Merkel and Langerhans cells,
later join in to form a community of specialized cells that together perform a
unique set of physiological functions. Despite their shared origin, hair
follicles and epidermal keratinocytes diverge to execute unique
differentiation programs and, in the adult, replenish themselves from distinct
stem cell populations (Alonso and Fuchs,
2003; Ito et al.,
2005). Epidermal stem cells are restricted to the innermost
(basal) layer and are the only epidermal cells that proliferate in the adult
under normal circumstances (Clayton et
al., 2007). Asymmetric basal cell divisions result in one cell
withdrawing from the cell cycle and commencing a terminal differentiation
program and a journey outwards towards the skin surface
(Lechler and Fuchs, 2005).
Differentiation is manifested by unique gene expression profiles that
distinguish the spinous layer, granular layer and cornified layer
(Fuchs and Raghavan, 2002;
Jamora and Fuchs, 2002).
In the hair follicle, keratinocytes surround a teardrop-shaped core of
mesenchymal cells (dermal papilla, DP) in a structure called the bulb. In
contrast to the epidermis, the proliferation zone within the bulb contains
many cells that are not in direct contact with a basement membrane, although
the lineage-restricted stem cells do maintain contact with the DP
(Legue and Nicolas, 2005). As
hair matrix keratinocytes exit the cell cycle, they differentiate into several
different cell layers [outer and inner root sheath, cortex and medulla
(Hardy, 1992;
Millar, 2002;
Sengel, 1976)]. In the third
week of life, murine hair follicles start to cycle (i.e. they undergo a period
of destruction, rest and regeneration), which continues throughout adult life
(Muller-Rover et al., 2001;
Stenn and Paus, 2001). The
hair shaft is actively produced during the anagen phase. During the
destructive catagen phase, the bottom two thirds of each hair follicle
undergoes apoptosis; only the upper third of the follicle, the stem cell niche
(bulge) and the DP remain intact. Catagen lasts for approximately 3 days and
is followed by the resting phase (telogen) of variable length. Subsequent
entry into anagen is believed to begin when signals from the DP activate stem
cells in the bulge (Stenn and Paus,
2001).
Notch signaling promotes selection of the hair fate by activated bulge stem
cells (Yamamoto et al., 2003)
and plays an important role in the maintenance of the follicle, in particular
in the inner root sheath fate (Pan et al.,
2004). Notch proteins (Notch1-4 in mice and humans) are
transmembrane receptors activated by transmembrane ligands on neighboring
cells (Kopan, 2002). Binding
of Notch to its ligand leads to its cleavage by 
-secretase, resulting
in translocation of the Notch intracellular domain (NICD) into the nucleus,
where NICD binds to CSL [named from the homologous proteins CBF1 (RBPj
,
Rbpj; Mouse), Su(H) (Drosophila) and LAG-1 (Caenorhabditis
elegans)]. CSL is a transcriptional repressor bound with other nuclear
factors to the promoter region of particular genes. Association of NICD with
CSL transiently converts CSL from transcription repressor to transcription
activator, resulting in activation of downstream targets
(Fryer et al., 2004;
Mumm and Kopan, 2000).
In the embryonic mouse epidermis, Notch1 intracellular domain (NICD1) is
ubiquitous in supra-basal keratinocytes selecting the spinous fate
(Blanpain et al., 2006;
Lin and Kopan, 2003;
Pan et al., 2004); after
birth, NICD1 is detected transiently in a small fraction of spinous cells (Y.
Pan and R.K., unpublished observations). This pattern of Notch1 activation is
consistent with its proposed role in suppressing proliferation and promoting
differentiation via cell-autonomous modulation of targets, which might include
Wnt, p21cip1 (Cdkn1a), K1 (Krt1), K10 (Krt10), Hes1 and p63 (Trp63)
(Blanpain et al., 2006;
Devgan et al., 2005;
Mammucari et al., 2005;
Nguyen et al., 2006;
Okuyama et al., 2004;
Rangarajan et al., 2001). In
addition, keratinocytes deficient in Notch1 are sensitive to chemical
carcinogenesis, establishing Notch1 as a tumor suppressor in the epidermis
(Nicolas et al., 2003).
Although the role of Notch2 in the epidermis is thought to be minimal
(Rangarajan et al., 2001),
loss of both Notch1 and Notch2, RBPj or presenilins (the catalytic
components of -secretase) results in more severe epidermal phenotypes
than loss of Notch1 alone (Blanpain et al.,
2006; Pan et al.,
2004), indicating that Notch2 also contributes to epidermal
differentiation.
The contribution of Notch signaling to follicular and epidermal homeostasis
can result from both cell-autonomous and cell non-autonomous effects. Both are
consistent with the role of Notch as a transcriptional modulator: the former
is built on the proven ability of Notch to modulate the transcriptional
landscape within keratinocytes (Blanpain et
al., 2006; Devgan et al.,
2005; Nguyen et al.,
2006), whereas the latter posits that some of the transcripts
altered by Notch encode cell surface or secreted molecules that would impact
neighboring cells (Lin et al.,
2000; Pan et al.,
2004). In this report, we demonstrate that Notch1 plays
contrasting roles in keratinocyte proliferation within the hair follicles and
the epidermis, attributed largely to previously underappreciated cell
non-autonomous signals. We provide evidence that Tgfß signaling is
elevated and that Kitl (Scf) expression is reduced in
Notch1-deficient hair matrix keratinocytes, correlating with reduced
keratinocyte mitotic rates and a reduced melanocyte population, respectively.
In addition, we demonstrate that the impact on the epithelial cell cycle is
not dependent on autocrine Tgfß reception in the keratinocyte but on
paracrine signaling to neighboring fibroblasts. The fibroblast, in turn,
controls keratinocyte proliferation by modulating insulin-like growth factor
(IGF) signaling: in the hair follicle, IGF binding protein 3 (Igfbp3)
levels were elevated in the DP, whereas, in the epidermis, Igfbp4
protein was reduced. Increasing the IGF/insulin-like growth factor binding
protein (IGFBP) ratio by Igf1 overexpression in Notch1-deficient skin restored
the cell numbers in the hair matrix. Thus, diffusible epithelial Tgfß and
stromal IGFBPs act downstream of Notch1 in a bi-compartmental signaling
network within the skin. When perturbed by the loss of Notch, this paracrine
loop could contribute to epidermal keratinocyte hyperproliferation in a manner
analogous to the vicious cycle seen in bone metastasis
(Mundy, 2002;
Roodman, 2004).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), Trp53-null (p53-null)
(Jacks et al., 1994),
Msx2-Cre (Sun et al.,
2000), Ivl::Igf1
(Weger and Schlake, 2005a) and
Notch1-conditional knockout (N1CKO)
(Msx2-Cre;Notch1flox/flox)
(Pan et al., 2004) mice were
described previously.
Notch1 Igf1 double mutants
Ivl::Igf1 females were crossed with N1CKO males, and the F1
Msx2-Cre/+;Notch1flox/+;Ivl::IGF1 male offspring were then
crossed with F1 Notch1flox/+;Ivl::IGF1 females to produce
Msx2-Cre/+;Notch1flox/flox;Ivl::IGF1 offspring.
Notch1 p53 (Trp53) double mutants
Female mice containing Trp53-/- were crossed with N1CKO
males, and the male Trp53-/- mice were crossed with
Notch1flox/flox females. The F1 male offspring
Msx2-Cre/+;Notch1flox/+;Trp53+/- were then
crossed with the F1 females
Notch1flox/+;Trp53+/-. The F2 offspring,
Msx2-Cre/+;Notch1flox/flox;Trp53+/- males and
Notch1flox/flox;Trp53-/- females, were mated to
produce the compound-mutant mice
Msx2Cre/+;Notch1flox/flox;Trp53-/- (abbreviated
to N1p53DCKO), which, along with their littermates, were used for
analysis.
Notch1 Tgfbr2 double mutants
Tgfbr2flox/flox males and females were similarly used
to generate
Msx2-Cre/+;Notch1flox/flox;Tgfbr2flox/flox
(abbreviated to N1TGFßR2CKO) for analysis.
PCR conditions and protocols used for genotyping will be provided upon request.
Histology, in situ hybridization (ISH) and immunohistochemical analysis
To obtain stereoscopic images, 9-day-old (P9) skins in PBS were torn as
strips across the caudal-rostral axis with fine forceps and photographed with
an Olympus SZH10 stereo dissecting microscope. For hematoxylin and Eosin
staining, ISH and immunohistochemical analyses, skins were fixed in 4%
paraformaldehyde in PBS, embedded in paraffin and sectioned at 5 µm
thickness. ISH was performed as described
(Pan et al., 2004) with probes
prepared using cDNAs generated by either reverse transcriptase (RT)-PCR or
from the following sources: Cdkn1a, IMAGE, 3495942; Nrarp,
IMAGE, 6439916; and Notch1 (Kopan
and Weintraub, 1993). The primer sequences for RT-PCR are
available upon request. For each reaction, a corresponding sense transcript
was used as a negative control. Immunohistochemical analysis was performed as
described previously (Lin et al.,
2000; Pan et al.,
2004). For BrdU staining, animals were intraperitoneally
administered with 100 µg BrdU/g body weight and sacrificed after 30
minutes. Immunohistochemical analyses with AE13, BrdU and Ki67 antibodies were
performed as described (Lin and Kopan,
2003; Lin et al.,
2000). Other antibodies used were Hes1 (rabbit, 1:1000; kindly
provided by T. Sudo, Toray Industries, Kamakura, Japan)
(Ito et al., 2000), Igfbp2
(goat, 1:200; R&D Systems, AF797), Igfbp3 (goat, 1:200; R&D Systems,
AF775) and p73 (mouse, 1:1000; Labvision, ER15). For ß-galactosidase
(rabbit, 1:1000, 5 Prime
3 Prime) and Dct (goat, 1:200, Santa Cruz,
sc-10451)-antibody staining, skins were fixed in
periodate-lysine-paraformaldehyde, cryoprotected with 30% sucrose in PBS
overnight, embedded in OCT and sectioned at 7 µm thickness. Sections were
counterstained with DAPI.
Cell counting and progenitor cell labeling index
For hair matrix cell counting, paraffin sections were stained with DAPI and
photographed under both UV and visible light. The two pictures were overlapped
and nuclei located below the most proximal melanin granules were counted from
each hair follicle. For each genotype, >20 hair follicles in proper
longitudinal orientation were counted to calculate average and standard
deviation. For progenitor cell labeling index, BrdU- and Ki67-positive cells
were counted from >20 hair follicles per mouse, using three mice for each
genotype. For calculating the melanocyte cell fraction, the number of
Dct-positive cells was divided by the number of hair matrix cells in each
follicle. The average percentage and standard deviation from >20 hair
follicles per genotype was calculated. In all cases, significant differences
between means were determined by the two-tailed Student's t-test.
TUNEL staining
Frozen unfixed dorsal skins were embedded in OCT, and 10 µm sections
were prepared for TUNEL (terminal deoxynucleotide transferase dUTP-digoxigenin
nick end labeling) assay. TUNEL assay was performed as described
(Lindner et al., 1997). The
number of TUNEL-positive cells per follicle was counted from >60
longitudinally sectioned, properly oriented follicles from each individual
[anagen-deleted Notch1 (anN1) and embryo-deleted Notch1 (emN1) follicles from
N1CKO mice at P9; wild-type follicles at P9 and P16], and three different
individuals were used. Statistical significance was determined by the
two-tailed Student's t-test.
Isolation of hair bulbs and epidermis, total RNA extraction, and microarray analyses
For hair bulb isolation, fresh P9 skin samples were immediately immersed in
RNAlater (Ambion, TX) and individual hair follicles were collected using a
fine tungsten needle and precisely cut with a 26G syringe needle (Becton
Dickinson and Company, NJ) at the level twice the distance between the tip of
the bulb and the melanin granules (Fig.
3A). For epidermis preparation, a patch of P9 dorsal skin was
harvested and flash-frozen with dry ice, and the epidermis was isolated by
scraping it off the skin using a cold scalpel. Isolated hair bulbs and
epidermis samples were then subjected to total RNA isolation using QIAGEN
RNeasy micro kit (QIAGEN Sciences, MD) according to the manufacturer's
protocol. For hair bulb microarray analyses, 50-100 ng of total RNA was
collected from emN1 and anN1 follicles isolated from the same N1CKO mouse
(total of two mice) and from two wild-type littermates. After double-round
amplification/labeling procedures, the cRNA products were hybridized to
Affymetrix MOE430v2 chips. All protocols were conducted at the Washington
University Genechip Facility
(http://www.siteman.wustl.edu/internal.aspx?id=238).
Affymetrix micro - array suite 5.0 and dChip software
(http://www.dchip.org)
were used for expression profile comparisons and GO (gene ontology)-based
classification. The data discussed in this publication have been deposited in
NCBIs gene expression omnibus (GEO,
http://www.ncbi.nlm.nih.gov/geo/)
and are accessible through GEO Series accession number GSE6867. Ingenuity and
David2
(http://niaid.abcc.ncifcrf.gov/)
were used for pathway analyses.
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CT) method. The histograms in the
figures show the mean±s.e.m. of two or three independent experiments
for hair follicles and of five independent experiments for epidermal samples,
each of which was performed in triplicate. All qRT-PCR primers were tested for
the presence of non-specific primer dimers and for linear amplification. The
identity of the qRT-PCR amplicons was validated by DNA sequencing or
restriction-enzyme digestion of the amplicon. Primer sequences and qRT-PCR
conditions are available upon request.
Immunoblotting
Frozen epidermal samples were immediately lysed in NP40 lysis buffer (1%
NP-40, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 20 mM Tris, pH 8.0) containing
protease and phosphatase inhibitors and analyzed by SDS-PAGE/western blotting.
The following primary antibodies were used: 
-tubulin (mouse, 1:1000;
Sigma-Aldrich B-5-2), cyclin D1 (rabbit, 1:1500; Santa Cruz sc-718), Igfbp4
(rabbit, 1:5000; kindly provided by Chernausek, Cincinnati Children's
Hospital, Cincinnati, OH), p15 (Cdkn2b; rabbit, 1:200; Santa Cruz sc-612), p21
(mouse, 1:100; Santa Cruz sc-6246) and anti-Tgfß2 (rabbit, 1:200; Santa
Cruz sc-90). Western blot quantification was performed using Quantity One
software (Bio-Rad Laboratories, CA). The histograms in the figures show
mean±s.e.m. of two or three independent epidermal lysates per
genotype.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). As the names imply, emN1 epidermis and follicles are
derived from cells that experienced a pulse of Cre activity at embryonic day
(E)9.5 and thus lack Notch1 from that point onwards. Conversely, anN1
follicles express Cre recombinase in the mid-bulb region a few days after the
start of the growth phase (anagen); Notch1 is thus only removed in the upper
bulb (Fig. 1A). We observed
that emN1 follicles were substantially shorter than anN1 or wild-type
follicles (Fig. 1B,C). Because
anN1 follicles were wild type in appearance, we concluded that growth-related
Notch1 functions are required in the proximal, actively proliferating matrix
during hair morphogenesis (Fig.
1B,C) (Pan et al.,
2004).
Closer inspection of hair bulbs at postnatal day (P)9 revealed that the
portion below the most-proximal melanin granules (marked by a dashed red line
in Fig. 1D) was shorter in the
emN1 follicles compared with wild-type or anN1 follicles
(Fig. 1D). Cell size in all
genotypes was not altered. Instead, fewer cells were detected in
Notch1-deficient follicles: emN1 hair follicles had only 44.69±8.92
cells, whereas wild-type (75.32±10.30) and anN1 (69.86±14.17)
hair follicles had nearly twice as many cells
(Fig. 1E). Notch1-deficient
epidermis did not display any observable hyperplasia at this age, but the
epidermis of older mice did (Fig.
1F,G) (Nicolas et al.,
2003; Rangarajan et al.,
2001). We thus conclude that, in contrast to the epidermis, in
which the number of Notch1-deficient epidermal keratinocytes increases as mice
age (Nicolas et al., 2003;
Rangarajan et al., 2001),
fewer Notch1-deficient keratinocytes formed the hair bulb. Therefore, Notch1
loss leads to differential effects on related keratinocyte populations.
|
;
Lindner et al., 1997;
Muller-Rover et al., 2001;
Weedon and Strutton, 1981).
One trivial explanation for reduced keratinocyte numbers in emN1 bulbs
compared with wild type is accelerated catagen onset, as observed in mice with
global epithelial deletion of Notch1 [K14-Cre;
Notch1flox/flox and K5-CreERT;
Notch1flox/flox
(Vauclair et al., 2005)]. To
determine whether reduced cell numbers in emN1 hair bulbs reflected early
catagen onset at P9, we examined emN1 skin at P9, P13, P17 and P22
(Fig. 2A). If catagen had
started at P9, the lower two thirds of the follicles would have been lost by
P13. Instead, characteristic follicular catagen destruction was not detected
before P17 in all genotypes, indicating that Notch1-deficient skin in N1CKO
mice did not exhibit accelerated entry into catagen
(Fig. 2A).
Early catagen (stage II) follicles contain, on average, four or more
TUNEL-positive cells (Lindner et al.,
1997) (Fig. 2B,C).
Although we observed a few Notch1-deficient follicles containing more than two
TUNEL-positive cells, which is characteristic of the morphologically distinct
catagen stage I (Muller-Rover et al.,
2001), more than 40% of emN1 hair follicles lacked TUNEL-positive
cells. Moreover, the average number of TUNEL-positive cells was significantly
lower in emN1 follicles at P9 compared with that seen at P16, at which point,
based on morphology, the cells are already undergoing catagen (1.34 versus
4.18, respectively; P<0.005;
Fig. 2C). Given that no further
morphological changes were detected, we concluded that the increased apoptotic
cell death in emN1 follicles at P9 did not indicate catagen onset nor could it
explain the reduced cell number seen in TUNEL-negative emN1 hair
follicles.
Another possible explanation for the lower cell numbers in emN1 follicles
is a reduced proliferation rate. To examine mitotic rates at P9, we labeled
mitotic cells with Ki67 (Fig.
2D, red) and S-phase cells by BrdU-incorporation
(Fig. 2D, green) following a
30-minute BrdU pulse-label (Chenn and
Walsh, 2002). The proportion of S-phase cells in the cycling
population (BrdU+/Ki67+) was determined for the hair follicles and the
epidermis of mutant and wild-type mice. Interestingly, emN1 hair follicles
displayed a reduced progenitor-labeling index (27.07±1.99%) compared
with anN1 (36.26±1.33%) and wild-type (35.28±0.71%) follicles
(Fig. 2E), which correlated
with the cell numbers in these follicles. Therefore, the lower cell numbers
within Notch1-deficient hair bulbs is probably due to a delayed cell cycle,
with a small contribution from increased apoptotic cell death. Interestingly,
P9 epidermal keratinocytes did not display any difference in the
progenitor-labeling index (Fig.
2E), indicating that the proliferation defect in epidermal
keratinocytes is progressive, evident only as the animals age.
|
;
DiSalvo et al., 1995;
Malkinson and Keane, 1978).
The reduced mitotic rate in emN1 follicles might be attributable to changes in
G1 length or in the G1/S checkpoint, perhaps due to decreased
expression/stability of G1 cyclins or increased levels of cyclin kinase
inhibitors (CKIs) (Mainprize et al.,
2001; Massague,
2004; Sherr and Roberts,
1999). To address the molecular mechanism underlying the lowered
mitotic index and increased apoptosis seen in Notch1-deficient hair follicles,
we performed microarray analyses on total RNAs extracted from a pool of
approximately 100 micro-dissected emN1 and anN1 hair bulbs (isolated from the
same N1CKO mouse) as well as wild-type hair bulbs isolated from a littermate
(Fig. 3A). emN1 and anN1
samples from one animal were compared to each other and to a wild-type
littermate in silico; differences present in two biological duplicates were
then compiled to produce a `proximal bulb' signature (the precise comparison
matrix of the six samples is available upon request). Genes altered in the
emN1 bulb but not in the anN1 bulb or in the anN1/wild-type comparison were
subjected to GO (gene ontology)-based classification and Ingenuity pathway
analysis. We identified 47 genes altered in Notch1-deficient hair matrix that
are involved in cell growth, cell cycle, cell proliferation or programmed cell
death (see Table S1 in the supplementary material).
Microarray analysis and quantitative reverse transcriptase (qRT)-PCR
revealed that transcription of the CKIs Cdkn1a (encoding
p21Cip1), Cdkn2b (encoding p15INK4b) and
Cdkn1c (encoding p57Kip2) was increased upon Notch1 loss,
whereas mRNA expression of the G1 cyclins (cyclin D1 and cyclin E) did not
change (Fig. 3B and see Table
S1 in the supplementary material). In addition, the expression of
Gsdm1 (gasdermin 1), thought to promote cell cycle arrest in
keratinocytes and in the gastrointestinal tract
(Lunny et al., 2005;
Saeki et al., 2000), was
significantly increased (Fig.
3B and see Table S1 in the supplementary material). Moreover, the
mRNA level of a proapoptotic gene, Scotin, was also significantly
induced upon Notch1 loss (Fig.
3B), consistent with the observed increase in apoptosis
(Fig. 2C). These data are
consistent with the hypothesis that loss of Notch1 affected the length of the
G1 phase via the increased expression of cytostatic genes and caused a slight
increase in apoptosis due to elevated levels of proapoptotic gene expression
in follicular keratinocytes.
|
;
Rossi et al., 2004;
Terrinoni et al., 2004) and
Igfbp3 (Fig. 5A,
described in detail below). Members of the p53 family are known regulators of
G1/S arrest and of apoptosis (Bartek and
Lukas, 2001; Massague,
2004; Moll and Slade,
2004). In the epidermis, loss of Notch1 resulted in elevated p63
levels (Nguyen et al., 2006),
which in turn contributed to proliferation at the expense of differentiation.
Trp63 (encoding p63) transcripts were slightly elevated in
Notch1-deficient follicles (Fig.
4A), whereas transcripts of Trp73 (encoding p73) were
greatly elevated (Fig. 4A). In
wild-type mice, p73 expression was restricted to a subset of
lineage-restricted stem cells adjacent to the DP
(Fig. 4B). In the absence of
Notch1, p73 expression extended to inner root sheath and outer root
sheath cells (Fig. 4B),
indicating that Notch1 or its targets might negatively regulate p73
expression. However, this expression pattern did not overlap with the
expression domain of any of the p53 family targets
(Fig. 4B,
Fig. 5B and
Fig. 8B). Moreover,
p73-positive cells were actively cycling, as evident from BrdU-incorporation
studies (compare Fig. 2D with
Fig. 4B).
Notch loss can lead to a reduction in p53 activity in neurons or cultured
cells (Huang et al., 2004;
Yang et al., 2004). To explore
whether changes in p53 accumulation contributed to the
Notch1-/- phenotype, or enhanced it, we crossed our N1CKO
mice with p53-deficient mice. Skin morphology and histology of compound
homozygous mice
(Msx2-Cre;Notch1flox/flox;Trp53-/-) were
indistinguishable from that of N1CKO (see Fig. S1 in the supplementary
material). Importantly, the mRNAs of the p53 family targets Igfbp3,
Cdkn1a and Scotin did not change in compound
Msx2-Cre;Notch1flox/flox;Trp53-/- hair
follicles (Fig. 4C). Therefore,
we conclude that none of the p53 family members contribute significantly to
the Notch1-/- phenotype in the hair follicle.
Altered IGF signaling in Notch1-deficient hair follicles
Our microarray analyses revealed that transcripts for IGF signaling
modulators (Igfbp2, Igfbp3 and Igfbp4) are elevated in Notch1-deficient hair
bulbs, as is the IGF receptor (Fig.
5A and see Table S1 in the supplementary material). Indeed,
pathway analysis of transcripts altered in emN1 relative to wild-type
follicles confirmed that the vast majority of possible protein networks were
anchored around IGFBPs (data not shown). IGF signaling promotes cell
proliferation by reducing the length of the G1 phase
(Edmondson et al., 2003), the
inverse of the phenotype we observed in Notch1-deficient hair follicles
(Fig. 2). Diffusible IGFBPs
antagonize the mitogenic (Baserga et al.,
1997; Firth and Baxter,
2002) and anti-apoptotic (Butt
et al., 1999; Resnicoff and
Baserga, 1998) activity of Igf1 by sequestering it from its
receptors (Edmondson et al.,
2003; Firth and Baxter,
2002).
To determine whether changes in IGF signaling underlie the Notch1-/- phenotype, we attempted to confirm the observed changes in IGFBP expression in the microarray at the tissue level. Igfbp2 expression was detected in the nuclei of hair matrix cells, and no change in its distribution was evident at the protein level in Notch1-deficient hair follicles (Fig. 5B). Strikingly, whereas Igfbp3 mRNA and protein were undetectable in wild-type hair follicles, their expression was evident in DP fibroblasts but not the keratinocytes of Notch1-deficient hair follicles (Fig. 5B).
Igfbp3 is a diffusible molecule and its ectopic expression in hair
follicles leads to decreased hair length and bulb volume
(Weger and Schlake, 2005b), a
phenotype strikingly similar to Notch1-deficient hair follicles (Figs
1 and
2). Conversely, ectopic
expression of Igf1 promotes hair growth and follicular elongation
(Su et al., 1999a;
Su et al., 1999b;
Weger and Schlake, 2005a).
Importantly, mice overexpressing both Igf1 and Igfbp3 are normal
(Weger and Schlake, 2005a),
indicating that IGF/Igfbp3 balance is an important regulator of follicular
keratinocyte proliferation.
To determine whether Igf1 overexpression (and thus a higher IGF/IGFBP
ratio) can rescue Notch1-deficient hair follicles, we generated
Msx2-Cre;Notch1flox/flox;Ivl::IGF1 mice
(Weger and Schlake, 2005a) and
analyzed the morphology of their hair follicles at P9. The cell numbers in
Notch1-deficient Igf1-expressing hair follicles increased
(Fig. 5C,D), indicating that an
imbalance between IGF ligand and IGFBPs was indeed a contributing factor to
the phenotype of Notch1-deficient follicles.
|
|
;
Jetten et al., 1986;
Moustakas et al., 2002), were
also altered in N1CKO skin. Transcription of the ligands Tgfb1 and
Tgfb2, the receptor Tgfbr2 [itself a target of Tgfß
signaling (Cui et al., 1995)],
the co-receptor Tgfbr3 (Brown et
al., 1999), as well as the Tgfß targets Col1a2
(Inagaki et al., 1994) and
Thbs1, were all elevated in the emN1 hair bulb
(Fig. 6A and see Table S1 in
the supplementary material). The ligands Tgfß1 and Tgfß2 and the
receptors TgfßRI and TgfßRII were all expressed in the hair follicle
(see Fig. S2 in the supplementary material)
(Paus et al., 1997). These
receptors were also expressed in the DP (see Fig. S2 in the supplementary
material), whereas Col1a2 was specifically expressed in dermal
fibroblasts (Zheng et al.,
2002). Because Tgfß-mediated signaling can act both as an
autocrine and a paracrine signal, this pathway is capable of activating the
transcription of Igfbp2 in keratinocytes
(Moustakas et al., 2002;
Scandura et al., 2004), as
well as that of Igfbp3 in neighboring cells
(Guo et al., 1995;
Martin and Baxter, 1991). To determine whether autocrine reception of Tgfß plays a role in hair matrix homeostasis, we removed the essential type II receptor Tgfbr2 in all Notch1-deficient keratinocytes by generating Msx2Cre;Tgfbr2flox/flox (termed TgfßR2CKO) and Msx2Cre;Notch1flox/flox;Tgfbr2flox/flox (termed N1TgfßR2CKO) mice. Only dermal fibroblasts and DP cells in these compound mice retained the ability to respond to Tgfß ligands via TgfßRII, yet the skin of TgfßR2CKO mice was indistinguishable from wild type (data not shown and see Fig. S3 in the supplementary material). Importantly, hair follicles from N1TgfßR2CKO and N1CKO mice were morphologically identical, and qRT-PCR analysis confirmed that the expression of Tgfb1, Tgfb2, Tgfbr3, Igfbp2, Igfbp3, Col1a2 and CKIs remained elevated in N1TgfßR2CKO emN1 hair bulbs (Fig. 6B). These observations indicate that: (1) even though a block in Tgfß signaling in bulb keratinocytes has not been demonstrated, TgfßRII-mediated Tgfß signals do not regulate the proliferation/apoptosis of hair matrix keratinocytes cell-autonomously; and (2) elevated Igfbp2 or CKI mRNA levels might not be a consequence of autocrine Tgfß signaling in the hair matrix. Collectively, our results described thus far are consistent with the hypothesis that Notch1 regulates hair matrix proliferation both autonomously, by preventing expression of proapoptotic and cytostatic genes, and non-autonomously, by establishing a bi-compartmental feed-back loop. Loss of Notch1 in transit amplifying keratinocytes induced the expression of Tgfß ligands, which were recognized by DP cells. As a possible consequence of Tgfß signaling, diffusible Igfbp3 was produced in DP cells to inhibit follicular IGF signaling and suppress the proliferation of follicular keratinocytes.
Bi-compartmental signals also impact melanocyte populations
In silico comparison of transcripts from Notch1-deficient and wild-type
hair bulbs revealed that expression of the diffusible factor Kitl
(Scf) was also significantly downregulated in Notch1-deficient hair
bulbs (Fig. 6C and see Table S1
in the supplementary material), suggesting that Notch1 is a positive regulator
of this melanocyte survival factor in hair matrix cells. Indeed, multiple
melanocyte signature transcripts were reduced (see Table S2 in the
supplementary material) (Rendl et al.,
2005), accompanied by a significant reduction in the number of
melanocytes [identified by Dct immunohistochemistry] in Notch1-deficient hair
bulbs (Fig. 6D,E), even after
correcting for total keratinocyte numbers. Notch-mediated activation of Hes1
contributes autonomously to melanoblast survival
(Moriyama et al., 2006);
however, Hes1 expression was not compromised in the melanocytes of N1CKO skin
(Fig. 8B and see Fig. S4 in the
supplementary material). Therefore, Kitl-dependent proliferation and
survival of follicular melanocytes
(Botchkareva et al., 2001;
Ito et al., 1999) is probably
dependent on Notch1 activity in keratinocytes.
|
;
Lamar et al., 2001) were
reduced in the epidermis and in the hair follicle
(Fig. 7C and
Fig. 8A). Transcripts of these
targets overlapped with Notch1 in the hair follicle
(Fig. 8B). By contrast,
epidermal Hes1 and p21Cip1 protein levels were reduced in
Notch1-deficient epidermal keratinocytes
(Fig. 7A,B), whereas their
expression was unchanged in Notch1-deficient hair follicles
(Fig. 3B and
Fig. 8). Moreover, the location
of expression of the Hes1 protein, a well-studied Notch target, did not
overlap with that of Notch1 mRNA
(Fig. 8B) or protein (see Fig.
S6 in the supplementary material). Instead, it overlapped with the expression
domain of Notch2 and Notch3 (for details, see
Pan et al., 2004). The
expression domain of Cdkn1a mRNA overlapped with that of the Hes1
protein, supporting an indirect activation mechanism whereby Hes1 impacts the
calcineurin (Ppp3ca)/nuclear factor of activated T-cells (NFAT) pathway
(Mammucari et al., 2005). We
conclude that p21Cip1 is probably a target of Hes1 (but not of
Notch1) in both the epidermis and the hair follicle, and that Hes1 acts as a
Notch1 target only in epidermal keratinocytes. In the hair follicle, its
expression might be regulated by other Notch paralogs (Notch2 or Notch3).
These data agree with a recent demonstration that vertebrate cellular context
affects the usage of targets by individual Notch proteins in a manner yet to
be explained (Cheng et al.,
2007). These differences constitute a cell-autonomous contribution
to the distinct proliferative behavior of hair follicles and epidermal
keratinocytes in response to Notch1 loss. |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Lin and Kopan, 2003;
Lin et al., 2000;
Pan et al., 2004). Although
several diffusible molecules, including Wingless and Unpaired, have been
identified as mediators of such cell non-autonomous Notch functions in
Drosophila (Giraldez and Cohen,
2003; Vaccari and Bilder,
2005), Kitl and Tgfß are the first mammalian factors
mediating bi-compartmental, Notch1-dependent signals that regulate
proliferation or survival of a neighboring cell population (e.g. melanocytes
within the hair follicles) to be identified. Nonetheless, reduced autocrine
Kitl does not contribute to the Notch1-deficient follicular phenotypes,
because animals treated with ACK2 (anti c-Kit antibody) or animals with
mutations that reduced Kitl levels do not display any phenotypes reminiscent
of Notch1 loss in their hair follicles
(Besmer et al., 1993;
Botchkareva et al., 2001;
Lecoin et al., 1995;
Okura et al., 1995). To our
knowledge, this is also the first report to place Notch1 upstream of Tgfß
signaling in any cell type. Although, at this time, we can only speculate as
to how Tgfß ligands responded to Notch1 loss, the broad spectrum of
co-regulated Tgfß response genes confirm that this pathway is perturbed
in response to Notch1 loss.
We observed a striking molecular difference between epidermis and hair
follicles in the response of the stromal compartment to Tgfß,
specifically, in regulating the expression of Igfbp3 and Igfbp4. In
Notch1-deficient hair follicles, induction of Igfbp3 underlies the follicular
phenotype in N1CKO animals. Overexpression of Igfbp4 can decrease the growth
of prostate cancer (Durai et al.,
2006); Igfbp4 mRNA was also up regulated in the follicle
(Fig. 5A) and might have also
contributed to the downregulation of Igf1. By contrast, Igfbp4 mRNA
was significantly downregulated in Notch1-deficient dermis
(Fig. 7A,B)
(Batch et al., 1994). We thus
posit that the differential modulation of IGF signaling as a consequence of
changes in stromal IGFBP levels might underlie the difference in the response
of epidermis and hair follicles to Notch1 loss. Therefore, the contribution of
Notch signaling to differential follicular and epidermal homeostasis results
from both cell-autonomous (differential target selection) and cell
non-autonomous (differential stromal response to the same diffusible factors)
effects, the latter mediated possibly by Tgfß
(Li et al., 2004;
Liu et al., 2001).
|
)]. However, catagen was not accelerated in N1CKO mice,
in which Notch1-deficient hair follicles resided in a heterogeneous
environment (i.e. in the presence of anN1 follicles, which have a wild-type
phenotype). Instead, synchronicity is maintained in the chimeric skin, with
Notch1-deficient follicles entering catagen along with the anN1 follicles. Our
studies provide mechanistic insight into the role of Notch1 in catagen
induction. Although several pro-catagen transcripts were elevated when Notch1 was lost, these changes were not sufficient to permit catagen onset in our chimeric N1CKO mice. For example, detection of Igfbp3 mRNA in N1CKO skin by in situ hybridization (ISH) required an extended incubation time in NBT reagent (48 hours) compared with signal development in early catagen follicles (3 hours; see Fig. S5 in the supplementary material). If catagen onset relied only on follicle-intrinsic mechanisms, it would be expected that Notch1-deficient follicles would enter catagen early, even if embedded in a heterogeneous environment. It is important to consider that, although our mice had chimeric skin, most individual follicles were not salt-and-paper mosaics; they either expressed Notch1 or they did not (Fig. 1A, Fig. 8B and see Fig. S6 in the supplementary material). This implies that the milder phenotype we see in Notch1-deficient follicles is not simply due to rescue by wild-type matrix cells.
Several other possible explanations for this observation exist. First, the
timing of Notch1 deletion in these experimental paradigms differs: dorsal
ectodermal Msx2-Cre expression
(Pan et al., 2004) precedes
dorsal keratin 14 (K14) expression
(Byrne et al., 1994); the
earliest catagen was induced by the postnatal Cre-mediated deletion [by
K5-CreERT in newborn mice
(Vauclair et al., 2005)].
Perhaps early deletion allowed for compensation by an intrinsic follicular
factor. Alternatively, accelerated catagen might be due, in part, to
Cre-mediated toxicity (Loonstra et al.,
2001; Schmidt et al.,
2000), which might be more severe with postnatal Cre induction.
However, we cannot rule out a third possibility: that the presence of
wild-type epidermis and follicles in Msx2-Cre lines ameliorates the
impact of Notch1 loss by the generation of systemic, diffusible factor(s) that
can entrain the cycling of Notch1-deficient follicles
(Stenn and Paus, 2001) and
prevent them from entering into catagen.
In conclusion, the response of wild-type stromal cells to signals produced
by mutant epithelial neighbors crucially contributes to cancer
(Krtolica and Campisi, 2002;
Krtolica and Campisi, 2003).
We demonstrate that Notch1 contributes to an elaborate network of signaling
pathways that monitor and control proliferation in the skin. The effects of
Notch1 loss in the skin are reminiscent of the transformation of cells
adjacent to Notch-deficient clones in Drosophila
(Moberg et al., 2005;
Thompson et al., 2005;
Vaccari and Bilder, 2005). Our
observations suggest that the skin tumorigenesis resulting from Notch loss is
in part mediated by elevation in the level of ligands that elicit stromal
responses, similar to the `vicious cycle' that promotes bone metastasis in
several solid cancers (Mundy,
2002; Roodman,
2004). In a broader context, the role of Notch signaling in tumor
suppression might reflect both an autonomous function in promoting
keratinocyte differentiation and a non-autonomous function within a signaling
network that regulates keratinocyte proliferation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/15/2795/DC1
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alonso, L. and Fuchs, E. (2003). Stem cells in
the skin: waste not, Wnt not. Genes Dev.
17,1189
-1200.
Bartek, J. and Lukas, J. (2001). Mammalian G1- and S-phase checkpoints in response to DNA damage. Curr. Opin. Cell Biol. 13,738 -747.[CrossRef][Medline]
Baserga, R., Hongo, A., Rubini, M., Prisco, M. and Valentinis, B. (1997). The IGF-I receptor in cell growth, transformation and apoptosis. Biochim. Biophys. Acta 1332,F105 -F126.[Medline]
Batch, J. A., Mercuri, F. A., Edmondson, S. R. and Werther, G. A. (1994). Localization of messenger ribonucleic acid for insulin-like growth factor-binding proteins in human skin by in situ hybridization. J. Clin. Endocrinol. Metab. 79,1444 -1449.[Abstract]
Besmer, P., Manova, K., Duttlinger, R., Huang, E. J., Packer, A., Gyssler, C. and Bachvarova, R. F. (1993). The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis. Dev. Suppl. 1993,125 -137.
Blanpain, C., Lowry, W. E., Pasolli, H. A. and Fuchs, E.
(2006). Canonical notch signaling functions as a commitment
switch in the epidermal lineage. Genes Dev.
20,3022
-3035.
Botchkareva, N. V., Khlgatian, M., Longley, B. J., Botchkarev,
V. A. and Gilchrest, B. A. (2001). SCF/c-kit signaling is
required for cyclic regeneration of the hair pigmentation unit.
FASEB J. 15,645
-658.
Brown, C. B., Boyer, A. S., Runyan, R. B. and Barnett, J. V.
(1999). Requirement of type III TGF-beta receptor for endocardial
cell transformation in the heart. Science
283,2080
-2082.
Butt, A. J., Firth, S. M. and Baxter, R. C. (1999). The IGF axis and programmed cell death. Immunol. Cell Biol. 77,256 -262.[CrossRef][Medline]
Byrne, C., Tainsky, M. and Fuchs, E. (1994).
Programming gene expression in developing epidermis.
Development 120,2369
-2383.
Cheng, H. T., Kim, M., Valerius, M. T., Surendran, K.,
Schuster-Gossler, K., Gossler, A., McMahon, A. P. and Kopan, R.
(2007). Notch2, but not Notch1, is required for proximal fate
acquisition in the mammalian nephron. Development
134,801
-811.
Chenn, A. and Walsh, C. A. (2002). Regulation
of cerebral cortical size by control of cell cycle exit in neural precursors.
Science 297,365
-369.
Clayton, E., Doupe, D. P., Klein, A. M., Winton, D. J., Simons, B. D. and Jones, P. H. (2007). A single type of progenitor cell maintains normal epidermis. Nature 446,185 -189.[CrossRef][Medline]
Cui, W., Fowlis, D. J., Cousins, F. M., Duffie, E., Bryson, S.,
Balmain, A. and Akhurst, R. J. (1995). Concerted action of
TGF-beta 1 and its type II receptor in control of epidermal homeostasis in
transgenic mice. Genes Dev.
9, 945-955.
Devgan, V., Mammucari, C., Millar, S. E., Brisken, C. and Dotto,
G. P. (2005). p21WAF1/Cip1 is a negative transcriptional
regulator of Wnt4 expression downstream of Notch1 activation. Genes
Dev. 19,1485
-1495.
DiSalvo, C. V., Zhang, D. and Jacobberger, J. W. (1995). Regulation of NIH-3T3 cell G1 phase transit by serum during exponential growth. Cell Prolif. 28,511 -524.[Medline]
Dry, F. W. (1926). The coat of the mouse (Mus musculus). J. Genet. 16,287 -340.
Durai, R., Davies, M., Yang, W., Yang, S. Y., Seifalian, A., Goldspink, G. and Winslet, M. (2006). Biology of insulin-like growth factor binding protein-4 and its role in cancer (review). Int. J. Oncol. 28,1317 -1325.[Medline]
Edmondson, S. R., Thumiger, S. P., Werther, G. A. and Wraight,
C. J. (2003). Epidermal homeostasis: the role of the growth
hormone and insulin-like growth factor systems. Endocr.
Rev. 24,737
-764.
Firth, S. M. and Baxter, R. C. (2002). Cellular
actions of the insulin-like growth factor binding proteins. Endocr.
Rev. 23,824
-854.
Fryer, C. J., White, J. B. and Jones, K. A. (2004). Mastermind recruits CycC:CDK8 to phosphorylate the notch ICD and coordinate activation with turnover. Mol. Cell 16,509 -520.[CrossRef][Medline]
Fuchs, E. and Raghavan, S. (2002). Getting under the skin of epidermal morphogenesis. Nat. Rev. Genet. 3,199 -209.[CrossRef][Medline]
Giraldez, A. J. and Cohen, S. M. (2003).
Wingless and Notch signaling provide cell survival cues and control cell
proliferation during wing development. Development
130,6533
-6543.
Guo, Y. S., Townsend, C. M., Jr, Jin, G. F., Beauchamp, R. D.
and Thompson, J. C. (1995). Differential regulation by
TGF-beta 1 and insulin of insulin-like growth factor binding protein-2 in
IEC-6 cells. Am. J. Physiol. Endocrinol. Metab.
268,E1199
-E1204.
Hardy, M. H. (1992). The secret life of the hair follicle. Trends Genet. 8, 55-61.[Medline]
Harms, K. L. and Chen, X. (2005). The C
terminus of p53 family proteins is a cell fate determinant. Mol.
Cell. Biol. 25,2014
-2030.
Huang, Q., Raya, A., DeJesus, P., Chao, S. H., Quon, K. C.,
Caldwell, J. S., Chanda, S. K., Izpisua-Belmonte, J. C. and Schultz, P. G.
(2004). Identification of p53 regulators by genome-wide
functional analysis. Proc. Natl. Acad. Sci. USA
101,3456
-3461.
Inagaki, Y., Truter, S. and Ramirez, F. (1994).
Transforming growth factor-beta stimulates alpha 2(I) collagen gene expression
through a cis-acting element that contains an Sp1-binding site. J.
Biol. Chem. 269,14828
-14834.
Ito, M., Kawa, Y., Ono, H., Okura, M., Baba, T., Kubota, Y., Nishikawa, S. I. and Mizoguchi, M. (1999). Removal of stem cell factor or addition of monoclonal anti-c-KIT antibody induces apoptosis in murine melanocyte precursors. J. Invest. Dermatol. 112,796 -801.[CrossRef][Medline]
Ito, M., Liu, Y., Yang, Z., Nguyen, J., Liang, F., Morris, R. J. and Cotsarelis, G. (2005). Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat. Med. 11,1351 -1354.[CrossRef][Medline]
Ito, T., Udaka, N., Yazawa, T., Okudela, K., Hayashi, H., Sudo, T., Guillemot, F., Kageyama, R. and Kitamura, H. (2000). Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium. Development 127,3913 -3921.[Abstract]
Jacks, T., Remington, L., Williams, B. O., Schmitt, E. M., Halachmi, S., Bronson, R. T. and Weinberg, R. A. (1994). Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4,1 -7.[Medline]
Jamora, C. and Fuchs, E. (2002). Intercellular adhesion, signalling and the cytoskeleton. Nat. Cell Biol. 4,E101 -E108.[CrossRef][Medline]
Jetten, A. M., Shirley, J. E. and Stoner, G. (1986). Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGF beta. Exp. Cell Res. 167,539 -549.[CrossRef][Medline]
Kopan, R. (2002). Notch: a membrane-bound
transcription factor. J. Cell Sci.
115,1095
-1097.
