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doi: 10.1242/10.1242/dev.00453
1 Program on Cell and Molecular Biology and Gene Therapy. CIEMAT, Avenue
Complutense 22, E28040 Madrid, Spain
2 Instituto de Biomedicina de Valencia. Jaime Roig 11, 46010-Valencia,
Spain
3 Department of Animal Pathology, Veterinary School, University of Santiago de
Compostela, E27002 Lugo, Spain
* Author for correspondence (e-mail: jesusm.paramio{at}ciemat.es)
Accepted 26 February 2003
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Np63 expression. These results indicate an essential role for p107 and
p130 in the epithelial-mesenchimal interactions.
Key words: Mouse, Keratinocytes, p107, p130
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Byrne et al., 1994;
Fuchs, 1998;
Fuchs and Byrne, 1994). Taken
together, the epidermis represents a perfectly suited model for studying
proliferation and differentiation. However, the molecular mechanisms governing
epidermal differentiation are still largely unknown.
The skin is also characterized by the presence of epidermal appendages.
Among them, the hair follicles are of a particular interest. These structures
develop through a series of epithelium-mesenchyme interactions (reviewed by
Fuchs, 1998;
Fuchs et al., 2001;
Fuchs and Raghavan, 2002;
Hardy, 1992;
Millar, 2002), which are
similar to those regulating the morphogenesis of other ectodermal organs such
as tooth (reviewed by Jernvall and
Thesleff, 2000). Among the different signals involved in hair
morphogenesis, recent experimental evidences have demonstrated that several
components of the Wnt pathway are essential. In fact, the downstream effectors
of Wnt signaling, ß-catenin and Lef1, are expressed in epithelial and
mesenchymal compartments as soon as hair follicle formation is induced
(Huelsken et al., 2001;
Zhou et al., 1995). In
addition, the specific ablation of the gene for ß-catenin in epidermis
results in a deficiency in hair follicle formation
(Huelsken et al., 2001),
whereas non-degradable ß-catenin expression in skin leads to de novo hair
formation (Gat et al., 1998).
Mice that lack Lef1, do not develop whiskers and show a reduced number of body
hairs (van Genderen et al.,
1994), and the increased expression of Lef1 in the epidermis of
transgenic animals leads to defects in the positioning and orientation of hair
follicles (Zhou et al.,
1995). Finally, the expression of dickkopf 1, a potent diffusible
inhibitor of Wnt action, in the skin of transgenic mice produces a complete
failure of placode formation prior to morphological or molecular signs of hair
differentiation (Andl et al.,
2002). In addition to Wnt, a number of other key signaling
pathways, including those modulated by fibroblast growth factors (FGFs)
(du Cros, 1993;
Ota et al., 2002;
Rosenquist and Martin, 1996;
Suzuki et al., 2000), bone
morphogenetic proteins (BMPs) (Blessing et
al., 1993; Botchkarev et al.,
2001; Botchkarev et al.,
1999; Botchkarev et al.,
2002; Kulessa et al.,
2000), TGFß (Foitzik et
al., 2000; Foitzik et al.,
1999; Paus et al.,
1997) and Shh (Bitgood and
McMahon, 1995; Chiang et al.,
1999; St-Jacques et al.,
1998), participate a reiterative manner during the hair follicle
development (reviewed by Millar,
2002). More recently, different members of the TNF
receptor
superfamily (Headon and Overbeek,
1999; Kojima et al.,
2000; Koppinen et al.,
2001; Laurikkala et al.,
2001; Laurikkala et al.,
2002; Mikkola et al.,
1999; Monreal et al.,
1999; Naito et al.,
2002; Schneider et al.,
2001; Thesleff and Mikkola,
2002) and subsequent signaling through NF
B family of
transcription factors (Schmidt-Ullrich et
al., 2001) have also been involved in hair follicle morphogenesis
and cycling. Finally, the transcriptional co-activator p63 (reviewed by
Brunner et al., 2002b;
Irwin and Kaelin, 2001;
Levrero et al., 2000;
Marin and Kaelin, 2000;
van Bokhoven and Brunner,
2002; Yang et al.,
2002; Yang and McKeon,
2000) is of crucial importance for correct development of
ectodermal appendages and mutations in the P63 gene (TP73L
– Human Gene Nomenclature Database) are found in a number of human
syndromes that are characterized by defects in hair and teeth (reviewed by
Brunner et al., 2002b;
van Bokhoven and McKeon,
2002). This protein, the most recently discovered member of the
p53 family, is expressed in embryonic ectoderm and in the basal, proliferative
layer of epidermis (Parsa et al.,
1999; Pellegrini et al.,
2001), and appears to be a keratinocyte stem cell marker
(Lee and Kimelman, 2002;
Pellegrini et al., 2001). In
agreement, besides other anomalies, the p63-knockout mouse lacks
epidermis, apparently owing to the loss of stem cells required for the tissue
(Mills et al., 1999;
Yang et al., 1999). However,
the actual molecular functions of p63 (Tcp1 – Mouse Genome Informatics)
in hair growth and development have not been yet delineated.
Cell cycle withdrawal is a common prerequisite for terminal differentiation
in most tissues. Consequently, those molecules implicated in cell cycle
regulation might have additional functions modulating cell differentiation.
This hypothesis has been clearly confirmed in the case of the retinoblastoma
family of proteins. This includes pRb, p107 (Rbl1 – Mouse Genome
Informatics) and p130 (Rbl2 – Mouse Genome Informatics). All these
proteins modulate cell cycle progression during G1 through their ability to
bind and inhibit different members of the E2f transcription factor family
(Classon and Dyson, 2001;
Weinberg, 1995), although
with different affinity, as E2f1-3 bind preferentially to pRb, whereas E2f4
and E2f5 bind to p107 and p130 (Classon
and Dyson, 2001). In addition, these proteins regulate different
aspects of the differentiation process in a number of tissues (reviewed by
Classon and Dyson, 2001;
Lipinski and Jacks, 1999), as
demonstrated by the analyses of mice lacking the different members of the
retinoblastoma family. Rb-deficient animals die between day 13 and 15 of
gestation, displaying overt defects in erithroid, neuronal and lens
development (Clarke et al.,
1992; Jacks et al.,
1992; Lee et al.,
1992). By contrast, mice deficient in p107 or p130 develop
normally and do not display any overt phenotype
(Cobrinik et al., 1996;
Lee et al., 1996), indicating
that, in most tissues, either p107 or p130 is dispensable for development, and
suggesting a functional overlap between them. In agreement, mice that lack
both proteins die immediately after birth and display defects in bone
development, associated with impaired chondrocyte differentiation
(Cobrinik et al., 1996).
However, Rbl1/p107-deficient embryos die earlier than their
Rbl1–/– littermates supporting the notion that
p107 can substitute some of the functions of pRb in differentiation, allowing
the extended survival observed in Rbl1-deficient embryos
(Lee et al., 1996).
Interestingly, the developmental consequences of p107 or p130 deficiency might
be determined by the genetic background
(LeCouter et al., 1998;
LeCouter et al., 1996).
With respect to the possible functions of the retinoblastoma family in
skin, we have reported that p107 and p130 are differentially expressed and
participate during in vitro human keratinocyte terminal differentiation
process (Paramio et al., 1998;
Paramio et al., 2000). To
investigate in more detail the relevance of the functionality of these
proteins in epidermal differentiation in vivo, we have analyzed the skin in
mice lacking p107 and/or p130. Our present results confirm that p107 and p130
are necessary to proper epidermal terminal differentiation and, in addition,
are essential mediators in the inductive signals between epithelium and
mesenchyme and important regulators of several morphogens involved in such
inductive interactions.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Lee et al.,
1996). Skin samples or whole embryos at 14.5, 16.5 and 18.5 dpc
(days post coitum) from wild-type and p107/p130-null mice were fixed in 10%
buffered formalin or 70% ethanol, and embedded in paraffin wax. Sections (5
µm) were stained with Hematoxylin/Eosin or processed for
immunohistochemistry and analyzed with a Zeiss Axiophot microscope.
Immunohistochemical methods
Immunohistochemistry was performed on deparaffinized sections using
antibodies against K10 (K8.60 mAb, 1/75 dilution, Sigma), loricrin (1/500
dilution of a monospecific rabbit polyclonal antibody, Covance) and filaggrin
(1/500 dilution of a monospecific rabbit polyclonal antibody, Covance).
Np63 was detected using 4A4 mAb (1/150 dilution; Santa Cruz
Biotechnology) in formalin-fixed samples. Bmp4 and noggin antibodies were
purchased from Santa Cruz Biotechnology (1/50 dilution), p75NTR (Ngfr –
Mouse Genome Informatics) from Covance (1/500 dilution) and Hgf from R&D
(1/50 dilution), and were used in formalin-fixed samples after microwave
treatment. The expression of EDAR and XEDAR (R&D) and TROY/TAJ (Santa Cruz
Biotechnology) (1/100 dilution) was monitored in ethanol fixed tissue
sections. Proliferation was monitored by PCNA staining using PC10 mAb
(generous gift of Dr D. P. Lane). p107 and p130 were detected in
formalin-fixed sections using specific rabbit polyclonal antibodies (1/150
dilution; Santa Cruz Biotechnology). Horseradish-labeled secondary antibodies
were purchased from Jackson Immunoresearch Laboratories and used 1:2000
dilution. The positive staining was visualized using Vector DAB kit, and
slides were counterstained with Hematoxylin. Negative control slides were
obtained by replacing the primary antibody with PBS (data not shown).
Northern blotting
Total RNA from frozen skin samples was isolated by guanidine
isothyocianate-phenol-chloroform extraction. Northern blots containing total
RNA (15 µg/lane) were probed for expression of the different signaling
molecules using full-length cDNA as probes. The membranes were also hybridized
with a 7S RNA probe to verify that equal amounts of mRNA were loaded and
transferred.
Mouse skin grafts
Dorsal full thickness skin pieces of 2-3 cm2 were obtained from
18.5 dpc p107/p130-null mice or double heterozygous littermates as control.
Donor skin pieces were grafted onto a wound created by removing a
similar-sized piece of full thickness back skin in female immunodeficient
NOD/scid (Prkdc – Mouse Genome Informatics) recipient
mice. Graft and host skin edges were joined using surgical silk suture and the
grafted area was covered with a thin layer of NewSkin (Medtech, Jackson) as
the only protective dressing. This procedure allows graft-take monitoring and
produces normal-haired donor skin. Graft recipient animals were routinely
monitored for hair growth and sacrificed 2-8 weeks after grafting. The
graft-containing area was excised and processed for histopathology or
immunohistochemical analysis as described above.
Band shift analysis
Electrophoretic mobility shift assays (EMSA) were performed by incubating
whole-cell extracts from mouse skin with a labeled oligonucleotide
corresponding to a palindromic B:
5'-GATCCAACGGCAGGGGAATTCCCCTCTCCTTA-3'.
Complexes were separated on 5.5% native polyacrylamide gels in 0.25x
Tris-borate-EDTA buffer, dried and exposed to Hyperfilm-MP (Amersham) at
–70°C. The composition of the B complexes in newborn mouse
skin has been previously described
(Budunova et al., 1999;
Perez et al., 2000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, no phenotypic alterations have been yet described
in vivo in the skin of these animals. Given the involvement of the Rb and E2f
families in differentiation of cultured human keratinocytes
(Paramio et al., 1998;
Paramio et al., 2000), we have
analyzed the epidermis of p107 and/or p130 knockout mice. No alterations were
found in the epidermis of animals lacking p107 or p130 compared with
heterozygous littermates (data not shown), indicating that, as in other
tissues, there exists compensation among the different members of the
retinoblastoma family (Cobrinik et al.,
1996). On the contrary, the epidermis of animals lacking both p107
and p130 displayed several abnormalities. We observed a reduction in the
number and size of keratohyalin granules suggestive of altered terminal
differentiation of the epidermal keratinocytes
(Fig. 1A,A',B,B').
To confirm this aspect we monitored the expression of several terminal
differentiation markers. We found a decreased expression of filaggrin
(Fig. 1D,D') and loricrin
(Fig. 1E,E') in
p107/p130-deficient epidermis. Conversely, we found that K10, an early marker
of the process, is expressed in the first suprabasal layer in the epidermis of
all the animals irrespective of their genotype
(Fig. 1C,C'). These
results indicate that, in agreement with our in vitro previous data
(Paramio et al., 1998), the
absence of p107 and p130 impairs the normal process of epidermal terminal
differentiation, but not the commitment of the process.
|
;
Paus et al., 1999). Based on
this classification, we also characterized a clear developmental delay in hair
follicle formation (Fig. 2E). This delay was evident as soon as day 14.5 dpc, at which the number of hair
germs was severely reduced (Fig.
2B,B'), and persisted at days 16.5 and 18.5 dpc (data not
shown and Fig. 2A,A'). In
mice, the pelage consists of different types of hair with different morphology
that are also formed at different times during embryogenesis. Primary or
tyolotrich (guard) are induced by day 14.5 dpc, whereas the morphogenesis of
secondary or nontylotrich (awl and zigzag) hair follicles starts by day 16.5
dpc. The fact that the differences between control and double-deficient mice
occur at these different embryonic stages
(Fig. 2D) strongly suggested
that the morphogenesis and development of both tylotrich and nontylotrich hair
follicles is affected in p107/p130-null skin. In addition, a similar delayed
development was also observed in the whiskers, specialized hairs of the murine
snout (Fig. 2C,C').
|
),
and in many cases the mutations that give rise to hair defects, such as those
in Lef1 (van Genderen et al.,
1994) or tabby-downless also display alterations in tooth
(Headon and Overbeek, 1999;
Laurikkala et al., 2001;
Laurikkala et al., 2002;
Miard et al., 1999;
Pispa et al., 1999;
Thesleff and Mikkola, 2002).
Consequently, we have analyzed the possible alterations in the development of
tooth germs in p107/p130-deficient mice. We commonly observed severe
alterations characterized by incisors microdontia
(Fig. 2G,G') and a few
cases of anodontia, which in the most extreme situation leads to the complete
absence of the incisors (arrows in Fig.
2F,F'). In addition, when present, tooth germs also
displayed severe histological defects such as hypoplasia of the odontoblast
layer (od in Fig. 2G,G'),
with poorly differentiated and disorganized cells (arrows in
Fig. 2G', compare with
2G).
Many of the alterations found in p107/p130-deficient mice were similar to
those found in mice bearing mutations in TNF-like superfamily mediated
signaling, including Eda-Edar, Xedar, Troy, etc.
(Headon and Overbeek, 1999;
Laurikkala et al., 2001;
Laurikkala et al., 2002;
Miard et al., 1999;
Pispa et al., 1999;
Thesleff and Mikkola, 2002)
or in the downstream NFB transcription factor family
(Schmidt-Ullrich et al.,
2001); consequently, we analyzed the expression of the receptors
Edar (Fig. 3A,A'), Xedar
(Fig. 3B,B') and Troy/Taj
(Fig. 3C,C') in control
and in p107/p130-deficient epidermis at 18.5 dpc. However, no differences were
observed between control and mutant hair follicles. In agreement, EMSA
analysis also showed similar endogenous NFB activity among the
different genotypes (Fig.
3D).
|
B-dependent signaling.
Hair formation in p107/p130-deficient skin transplants
The early death of p107/p130-deficient mice precludes the analysis of
epidermis and epidermal appendages in adults and the phenotypic evolution of
the observed defects. To avoid this problem, we grafted p107/p130 epidermis
onto NOD/scid mice. Hair growth in these transplants was evident four
weeks after grafting in control and in p107/p130-deficient transplants
(Fig. 4A,A',
respectively). Histological analysis demonstrated that, primary and secondary
hairs are formed in both (Fig.
4B; data not shown). In p107/p130-deficient grafts, most of the
hairs were morphologically normal with no alterations in any of the different
cell populations (Fig. 4B). In
addition, most anagen hair bulbs (Fig.
4C') were indistinguishable from those of controls
(Fig. 4C). However, a number of
several abnormalities were also frequently detected in p107/p130-deficient
transplants. First, we noticed that four weeks after grafting the number of
hairs (Fig. 5A,A')
exceeded that of controls (Fig.
5B) by an average of three times. In addition, a severe
interfollicular hyperplasia, which is in some areas associated to
parakeratosis, was observed in mutant transplants
(Fig. 5C). Secondly, besides
the formation of normal hairs, we also noticed multiple hair abnormalities
such as multiple hair follicles sharing a unique hair channel
(Fig. 5D), twisted hair
follicles lying in parallel to the epidermal surface
(Fig. 5E), multiple follicular
keratin-filled cysts (Fig. 5F),
and hyperplasic sebaceous glands (Fig.
5G). Finally, we also observed a major number of follicles in
anagen phase compared with controls (six times increase, on average). To
monitor if hair cycling was affected in p107/p130-deficient transplants, we
analyzed grafts at different times after transplantation. We observed that the
vast majority of hairs formed 2 and 3 weeks after transplantation were already
in anagen phase (Fig. 5H,I,
respectively); however, owing to the severe inflammation, which is associated
to the process of wound healing, we can not directly attribute this alteration
to intrinsic abnormalities. Conversely, we observed that 8 weeks after
grafting, while the vast majority of hairs in control samples were in resting
telogen phase (Fig. 5J); in
mutant samples almost all hairs were still in anagen phase
(Fig. 5K). Taken together,
these results indicate that, although the formation of normal hair occurs in
p107/p130 grafts, a number of abnormalities affecting hair morphogenesis and
development, as well as hair cycling, also arise in the absence of these
proteins.
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|
; Fuchs et al.,
2001; Fuchs and Raghavan,
2002; Hardy, 1992;
Millar, 2002). In control
skin, the expression of p107 and p130, besides interfollicular keratinocytes,
was detected in several cell types of the hair follicle, including the
epithelial outer and inner root sheaths and the cortex, as well as the
mesenchymal dermal papilla cells (Fig.
6A,B). Consequently, the hair developmental abnormalities of
p107/p130-deficient mice can thus be attributed to alterations in the
epithelial and/or in the mesenchymal compartments, as both are defective in
these proteins. However, graft experiments can also help to discriminate
between these possibilities. In fact, we observed that p107 and p130 proteins
were expressed in the dermal papilla of hair follicles in null grafts
(Fig. 6C,D), indicating that
fibroblasts from the recipient reconstituted a follicular dermal papilla
precursor, allowing the donor epithelium to develop into a mature anagen hair
follicle. Therefore, the hair follicle abnormalities found in mutant skin are
probably due to a defect in the mesenchyme that is restored in the
transplants.
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|
Altered expression of hair morphogens in absence of p107 and
p130
Among the different signals required for normal hair development are Shh
(Bitgood and McMahon, 1995;
Chiang et al., 1999;
St-Jacques et al., 1998) and
the components of the Wnt signaling pathway (reviewed by
Fuchs et al., 2001;
Fuchs and Raghavan, 2002;
Millar, 2002). We thus
monitored their expression by northern analyses. No major alterations were
found in the expression of Shh, ß-catenin, axin, Lef1 and Fwd1
(Fig. 8A; data not shown). In
addition, several members of the BMP/TGFß superfamily have been
demonstrated to modulate hair growth
(Blessing et al., 1993;
Botchkarev et al., 2001;
Botchkarev et al., 1999;
Botchkarev et al., 2002;
Foitzik et al., 2000;
Foitzik et al., 1999;
Kulessa et al., 2000;
Paus et al., 1997). An
important decrease in Bmp4 expression was observed in p107/p130-null samples
(Fig. 8A). To confirm this
observation further we analyzed the expression and localization of Bmp4
protein in control and p107/p130-deficient skin by 18.5 dpc. The expression of
Bmp4 in control skin (Fig. 8B)
is in accordance with previous reports
(Botchkarev et al., 2002;
Kulessa et al., 2000).
However, we failed to detect Bmp4 protein
(Fig. 8B') in the hair
follicles of the mutant skin, in agreement with the northern results. In hair
follicles, the Bmp4-dependent signaling is modulated by a number of different
morphogens, including noggin, p75NTR (Ngfr – Mouse Genome Informatics),
Lef1 and Hgf, whose expression is also affected by Bmp4
(Botchkarev et al., 2001;
Botchkarev et al., 1999;
Botchkarev et al., 2002;
Botchkareva et al., 1999;
Kulessa et al., 2000;
Lindner et al., 2000;
Zhao et al., 2000). We have
thus analyzed the expression and localization of these molecules in control
and in p107/p130-deficient hair follicles. We did not detect any significant
changes in p75NTR and Lef1 expression in mutant hair follicles
(Fig. 8C,C'; data not
shown). By contrast, the expression and localization of noggin and Hgf were
severely perturbed in mutant hair follicles when compared with controls
(Fig. 8D,D',E,E'). These results suggest that, in the absence of p107 and p130, there is a
defective Bmp4-dependent signaling that affects hair follicle morphogenesis
and development.
|
Decreased expression of Np63 in p107;130 deficient mouse
epidermis
We next investigated the possible consequences of the observed alteration
in Bmp4-dependent signaling. Among the possible targets of Bmp4 signaling,
Np63 has been recently identified in zebrafish as an ectoderm-specific
direct transcriptional target (Bakkers et
al., 2002). Moreover, two main aspects of p63 are of particular
interest for the present study. First, there is a clear association of
P63 mutations with different human syndromes characterized by altered
deficient hair and tooth growth (reviewed by
Brunner et al., 2002a;
Brunner et al., 2002b;
van Bokhoven and McKeon,
2002). Second, in normal epidermis and in hair follicles, the
Np63 variant is preponderant and appears restricted to cells with high
proliferative potential and is absent from the cells that are undergoing
terminal differentiation (Parsa et al.,
1999; Pellegrini et al.,
2001). Consequently, given the observed alterations in the
morphogenesis of hair and tooth (Fig.
2) and in keratinocyte proliferation
(Fig. 7), together with the
altered Bmp4 signaling (Fig.
8), it is possible to speculate that altered Np63
expression might occur in p107/p130 mutant skin. In control embryos at day
18.5, Np63 expression was observed in the nuclei of all basal cells in
the interfollicular epidermis and also in the rudimentary follicles
(Fig. 9A). Interestingly,
immunoreactivity in the positive cells is not homogeneous, as some cells
display increased Np63 (arrows in
Fig. 9A). In addition,
Np63 expression decreases as the cells enter the differentiation
program and migrate into the suprabasal layers
(Fig. 9A) as previously
reported (Parsa et al., 1999;
Pellegrini et al., 2001).
However, a severe reduction in the expression of Np63 was found in
p107/p130-deficient keratinocytes (Fig.
9B). Conversely, one would expect that, if the reduced Np63
expression was related to delayed development of the hairs in
p107/p130-deficient skin, such expression would be restored in the
transplants, given the restored hair growth observed in them. In fact,
immunohistochemical analyses corroborate such hypothesis as Np63 was
found to be normally expressed in the grafted skin
(Fig. 9C).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wang et al., 2001;
Wang et al., 2000), viral
oncogenes, such as HPV E7 (Greenhalgh et
al., 1994) or E1A (Missero et
al., 1993), or altered expression of cyclin-dependent kinase
inhibitors (Di Cunto et al.,
1998; Missero et al.,
1995; Paramio et al.,
2001) strongly support the existence of such roles. We have
previously shown that the three pocket proteins are functionally involved in
the process of in vitro differentiation of human keratinocytes, probably
through the interaction with specific E2f members
(Paramio et al., 1998;
Paramio et al., 2000). We have
focused on the roles of p107 and p130 in epidermis in vivo. We have found that
the absence of any of these molecules do not cause phenotypic alterations,
indicating that, as previously reported for other tissues
(Cobrinik et al., 1996), there
is functional compensation between these two proteins in skin. In agreement,
the simultaneous absence of p107 and p130 produces severe skin abnormalities
characterized by defects in epidermal terminal differentiation
(Fig. 1) and reduced number and
delayed morphogenesis of hair follicles
(Fig. 2). Finally, we also show
that the altered development of the hair follicle lies in defective
interactions between the epithelium and the mesenchyme that also affect other
organs requiring inductive interactions between these elements, such as
whiskers and tooth germs (Fig.
2).
In agreement with our previous results
(Paramio et al., 1998;
Paramio et al., 2000), we
found impaired terminal differentiation in absence of p107 and p130. At
present, we do not know the molecular basis for such alteration; however,
given that this seems to be a cell autonomous effect, as demonstrated in
epidermal grafts, it is possible to speculate that this is mediated by direct
interference with the epidermal differentiation program. In fact, several
epidermal-specific proteins are regulated by transcriptional elements such as
Sp1 or C/EBP (Byrne et al.,
1994; Kaufman et al.,
2002; Kumar and Butler,
1999; Maytin and Habener,
1998; Maytin et al.,
1999; Oh and Smart,
1998; Park and Morasso,
1999; Sinha and Fuchs,
2001; Zhu et al.,
2002), which are themselves modulated by retinoblastoma family
proteins (Charles et al.,
2001; Chen et al.,
1996; Classon et al.,
2000; Decesse et al.,
2001; Rohde et al.,
1996; Udvadia et al.,
1995). Conversely, we previously observed decreased E2f4
expression in terminally differentiating human keratinocytes, and most of the
protein is bound by p107 and p130 (Paramio
et al., 2000). Consequently, a possible explanation for the
observed alterations would be that E2f4 expression facilitates epidermal
differentiation commitment, whereas its expression at later stages would
inhibit the process. However, recent data suggest that the mechanisms acting
in mouse and human keratinocytes may differ, and complexes of E2f5 with p130
and histone deacetylases may be responsible for terminal differentiation of
cultured mouse keratinocytes (D'Souza et
al., 2001). The ongoing experiments will help to discern these
possibilities and to determine the possible causes of such altered
differentiation in epidermal keratinocytes as a consequence of p107 and p130
deficiency.
The altered development of the hair follicles and the incisors observed in
p107/p130-deficient mice is of a particular interest. To our knowledge, this
is the first evidence that these two proteins are involved in morphogenetic
events. However, this observation is not totally surprising as E2f proteins
mediate morphogenesis in Drosophila and Xenopus
(Asano and Wharton, 1999;
Hirose et al., 2001;
Page et al., 2001;
Suzuki and Hemmati-Brivanlou,
2000). More recently, the involvement of the retinoblastoma family
in the control of morphogenesis has been highlighted by the finding that these
proteins can form complexes with developmental factors that contain
paired-like homeodomains (Wiggan et al.,
1998). Interestingly, one of these factors, Alx4, displays an
expression pattern that is restricted to sites of epithelial-mesenchymal
interactions (Hudson et al.,
1998; Qu et al.,
1997a; Qu et al.,
1997b). The possible functional relationship between p107 and/or
p130 and Alx4 would merit future investigations.
The proper development of hair follicles and tooth require a large number
of sequential interactions between the epithelial and mesenchymal cells that
finally involve a tremendous plethora of different molecules belonging to
several interconnected pathways (reviewed by
Fuchs et al., 2001;
Jernvall and Thesleff, 2000;
Millar, 2002). The analysis of
the expression of some of such molecules revealed a decreased expression of
Bmp4 and altered Bmp4-dependent signaling
(Fig. 8), but not in Eda/edar
or in NFB-depedent signaling (Fig.
3). We also found that, besides Bmp4, p107/p130-deficient hair
follicles also displayed altered expression of noggin and Hgf, but not p75NTR
(Fig. 8). Although at present
we cannot rule out the possibility that these alterations, or those affecting
other pathways regulating hair morphogenesis, might be the responsible for the
findings in p107/p130-deficient mice, most of the phenotypic findings can be
explained in terms of such diminished Bmp4 signaling.
Mice that lack the BMP antagonist noggin
(Botchkarev et al., 2001;
Botchkarev et al., 1999;
Botchkarev et al., 2002)
demonstrated that unregulated BMP proteins inhibit secondary hair development,
show increased proliferation and reduced p75NTR, ß-catenin and Lef1
expression in hair follicles, and also show reduced keratin K10 expression in
the interfollicular keratinocytes. However, we observed that the absence of
p107 and p130 inhibits both primary and secondary hair follicle development,
without alterations in p75NTR, ß-catenin and Lef1 expression
(Fig. 8) and the effects on
keratin K10 were only detected in deficient grafts
(Fig. 7). Consequently, it is
possible to discard noggin as a mediator of the phenotypic alterations found
in skin of p107/p130-null mice. However, we found altered Hgf expression
(Fig. 8). Whether this
alteration might be upstream or downstream of Bmp4 signaling remains to be
elucidated. In this regard, it is worth mentioning that both proteins are
similarly expressed during Xenopus development
(Aberger et al., 1997), but
Bmp4, in contrast to Bmp2, cannot downregulate Hgf during limb bud
morphogenesis (Scaal et al.,
1999). The discrimination among these possibilities is a difficult
task primarily due to the early embryonic lethality displayed by Hgf-deficient
mice (Schmidt et al., 1995;
Uehara et al., 1995).
However, the fact that Hgf induces hair growth
(Lindner et al., 2000;
Shimaoka et al., 1995) would
support a possible role for Hgf in the hair phenotype of the p107/p130-null
mice. The functional relationship between BMPs and Hgf remains to be
elucidated.
Bmp4 appears to play a dual role as an inhibitor and also an activator of
hair and tooth development. Recent data also demonstrated that proper BMP
signaling is required for appropriate hair formation and development
(He et al., 2002;
Kulessa et al., 2000). In
agreement, Dlx3 and Msx2, which are activated by BMP signaling
(Park and Morasso, 2002), are
required for hair and tooth formation, and are associated with defective
endochondral bone formation (Satokata et
al., 2000). In this regard, it is worth mentioning that Bmp4
signaling is required for the maintenance of the differentiated postmitotic
status of chondrocytes during endochondral ossification
(Enomoto-Iwamoto et al., 1998;
Minina et al., 2001), a
process that is altered in p107/p130-null mice
(Cobrinik et al., 1996).
The apparent opposite functions of Bmp4 during hair and tooth development
might be due to different and sequential expression sites, either mesenchyme
or epithelium, observed during the development of these organs, and which
appear to be modulated by different transcriptional processes
(Feng et al., 2002;
Zhang et al., 2002). In
particular, the expression in the mesenchyme seems to be modulated by
sequences that contain putative sites for retinoblastoma and Sp1 binding
(Feng et al., 2002;
Zhang et al., 2002), which
may account for the observed altered expression. In this regard, although we
cannot discard that both the epithelium and the mesenchyme are responsible for
the hair phenotype, the fact that in mutant skin grafts hair growth
(Fig. 4) and the expression of
Bmp4, noggin and Hgf (Fig. 8)
are restored clearly points to mesenchymal defective signaling. This is
further supported by the finding that dermal papilla cells in the mutant
transplants express p107 and p130 (Fig.
6), suggesting that recipient fibroblasts have reconstituted a
follicular dermal papilla precursor that allows the donor epithelium to
develop anagen hair follicles.
Bmp4 signaling induces the expression of different target genes among which
Lef1 and Waf/Cip1 (Jernvall et al.,
1998; Kratochwil et al.,
1996) appears to be highly relevant for hair and tooth formation.
However, we do not detect decreased expression of any of these two genes in
p107/p130-null mice skin (Fig.
8 and data not shown). This is probably due to the fact that other
factors can compensate the decreased Bmp4 expression or, alternatively, that
such reduction is not enough to cause the repression of these genes. However,
our data are in agreement with the increased Lef1 and decreased Bmp4 gene
expression found using high-density oligonucleotide arrays to identify genes
changed in response to activation of E2f species
(Muller et al., 2001). More
recently, Np63 has been characterized as a transcriptional target of
Bmp4 signaling (Bakkers et al.,
2002). In agreement, we also detect decreased expression of
Np63 in the basal layer of the p107/p130-null epidermis
(Fig. 9). Mice that lack the
p63 gene, are devoid of most epidermis and epidermal derivatives
(Mills et al., 1999;
Yang et al., 1999), and
mutations in the P63 gene are found in a number of human syndromes
characterized by defects in hair and teeth (reviewed by
Brunner et al., 2002a;
Brunner et al., 2002b;
van Bokhoven and McKeon,
2002). The possible causal relationship between such reduced
Np63 levels and the hair phenotype will be the subject of future
investigation.
Collectively, we present here evidence for functional activities of the retinoblastoma family members in epidermis and epidermal appendages modulating not only differentiation and proliferation processes but also remarkably important morphogenetic events that lead to the formation of specialized ectodermal organs.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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