1 Cancer Research UK, London Research Institute, 61 Lincoln's Inn Fields, London
WC2A 3PX, UK
2 Cell Adhesion and Disease Laboratory, St Thomas Hospital, London SE1 7EH,
UK
Author for correspondence (e-mail:
clive.dickson{at}cancer.org.uk)
Accepted 4 August 2003
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SUMMARY |
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Key words: Fgfr2-IIIb, Skin, Epidermis, Hair follicle, Hair differentiation, Mouse
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Introduction |
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Fgfs are a large family of intercellular signalling molecules that mediate
their biological responses by binding and activating high affinity cell
surface receptors (Fgfr) (reviewed by
Johnson and Williams, 1993;
Ornitz et al., 1996
;
McKeehan et al., 1998
). There
are four genes that encode Fgf receptors with intrinsic tyrosine kinase
activity (Fgfr1 to Fgfr4), but additional complexity is
achieved by alternative splicing. Fgfr2 can encode two receptor
isoforms, Fgfr2-IIIb and Fgfr2-IIIc, that have different Fgf-binding
specificities and are expressed on different cell lineages. Fgfr2-IIIb is
located on the epithelia of ectodermal and endodermal organs, and is activated
by four known ligands, Fgf1, Fgf3, Fgf7 and Fgf10. The latter two Fgfs are
expressed predominantly in mesenchyme adjacent to epithelia expressing
Fgfr2-IIIb (Peters et al.,
1992
; Orr-Urtreger et al.,
1993
; Mason et al.,
1994
; Finch et al.,
1995
; Ornitz et al.,
1996
; Yamasaki et al.,
1996
). To understand the function of Fgf signalling in epidermal
and hair follicle development, we have examined in detail the phenotype of
mice deficient for Fgf10 or Fgfr2-IIIb.
The generation and characterisation of mice deficient for
Fgfr2-IIIb or Fgf10 has been described previously
(Revest et al., 2001;
Min et al., 1998
). The
Fgfr2-IIIb isoform-specific null mice were generated by placing translational
termination codons in three reading frames within exon IIIb, followed by an
IRES-lacZ cassette. These changes result in a truncated form of
Fgfr2-IIIb that lacks part of the ligand-binding domain, and the entire
transmembrane and tyrosine kinase domains. Expression of the alternatively
spliced isoform Fgfr2-IIIc remains intact. Previous studies have shown that
Fgfr2-IIIb is highly expressed in the basal keratinocyte layer
(Revest et al., 2001
;
Mailleux et al., 2002
). By
contrast, Fgf7 and Fgf10, the main ligands for this receptor, are expressed in
the dermis, consistent with a role in mesenchymal-epithelial signalling during
skin development (Mason et al.,
1994
; Beer et al.,
1997
; Mailleux et al.,
2002
). However, to date, the functional role of Fgf/Fgfr2-IIIb
signalling in epidermal and hair follicle development is not well
understood.
We show that mice null for Fgfr2-IIIb have a severe epidermal hypoplasia resulting from a loss of keratinocyte proliferative capacity. Despite the extremely thin suprabasal layer, epidermal differentiation and establishment of barrier function appears to proceed normally. Mice deficient for Fgf10 show a similar but less severe phenotype. Fgfr2-IIIb/ mice also show a reduced and abnormal development of hair follicles, a defect that is not recapitulated in the Fgf10/ mice. Transplantation of Fgfr2-IIIb/ skin also reveals abnormal hair type formation. A model to explain these finding is presented.
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Materials and methods |
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Histology, in situ hybridisation and immunohistochemistry
Histology and in situ hybridisation were carried out using standard
procedures and as previously described
(Revest et al., 2001). Probes
used were Bmp4, Shh and Lef1
(Revest et al., 2001
;
Mailleux et al., 2002
).
Wax sections of back skin were dewaxed in xylene, rehydrated through graded alcohols, and used for detection of loricrin (BabCO, USA) or Ki67 (Novocastra, USA). Sections were boiled twice for 10 minutes in 0.01 M citrate buffer (pH 6) then blocked with swine serum (1:25). All dilutions were in PBS. Anti-loricrin (1:250) or anti-Ki67 (1:200) was applied for 40 minutes at room temperature. A biotinylated swine anti-rabbit antibody (DAKO, UK) was applied (1:500) for 40 minutes, followed by streptavidin-peroxidase for 40 minutes (1:500). A DAB substrate kit (Vector Labs, USA) was used for detection.
Saggital wax sections of embryos were dewaxed in xylene, rehydrated through graded alcohols, and used for co-detection of BrdU and keratin 14 (K14). Sections were hydrolysed for 8 minutes in 1M HCl at 60°C, then blocked with goat serum (1:25). Anti-BrdU (Abcam, USA) and anti-K14 (BabCO, USA) antibodies were applied (1:1000) for 35 minutes at room temperature. A peroxidase-conjugated goat anti-rat antibody (BrdU) (1:200) and an alkaline phosphatase-conjugated goat anti-rabbit antibody (K14) (1:25) were co-incubated for 35 minutes. A DAB substrate kit (Vector Labs, USA) was used for detection of BrdU and an alkaline phosphatase kit (Vector Labs, USA) was used for detection of K14.
For immunofluorescence, cryosections were fixed for 10 minutes in cold acetone and blocked with goat serum (1:25). Anti-keratin 10 (Chemicon, UK)(1:500), anti-K14 and anti-nidogen (Chemicon, UK) (1:500) antibodies were co-incubated for an hour followed by goat anti-IgG1-TRITC (K10) and goat anti-rabbit-FITC with goat anti-rat-TRITC (K14 and nidogen, respectively) (1:100). Cell nuclei were labelled by using Hoechst stain (1:500) with the secondary antibodies.
Quantitative histomorphometry
The percentage of BrdU-positive cells in the basal layer of the
interfollicular epidermis was determined by counting three microscopic fields
on two slides from at least three different embryos at each stage of
development for both wild-type and
Fgfr2-IIIb/ mice. The thickness of the
epidermis was determined by measuring the length and area on the same slides
using NIH Image 1.61. The number of hair follicles per unit length of
epidermis, together with their morphological stage, was determined on the
basis of defined morphological criteria using Haematoxylin and Eosin stained
wax sections of E17.5 embryos (Hardy,
1992; Paus et al.,
1999
). Every tenth section from each specimen was analysed, thus
ensuring that the microscopic field contained new hair follicles. Five to 20
longitudinal sections per embryo were taken from the same anatomical site and
analysed: five Fgfr2-IIIb/ embryos
were compared with eight wild-type littermates; Fgf10 wild type
n=4, Fgf10/ n=4. All
sections were analysed at 100x magnification, means and s.e.m. were
calculated from pooled data. Differences were judged significant if
P<0.05, as determined by Student's t-test.
Skin permeability assay
Embryos were dissected, incubated for 1-5 minutes in methanol, rinsed in
PBS, and then incubated for 2 hours in 0.1% Toluidine Blue. Embryos were
rinsed in PBS and photographed (Hardman et
al., 1998).
Skin grafts
To perform skin grafts, a full-thickness skin disc (6 mm) was excised from
male nude mice. A similar biopsy punch from the back skin of E18.5 knockout
and wild-type littermate embryos was applied onto the recipient fascia.
Steri-strips, circular plasters and gauze bandages (Southern Syringe Service,
London, UK) were applied to secure the grafts in place. Dressings were removed
after 7 days. Grafts were excised and bisected along the anterioposterior axis
21 days after grafting. Half of each graft was fixed in 4% paraformaldehyde
overnight and processed for histological examination as above. Hairs were
plucked from the other half of the graft for microscopic analysis.
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Results |
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A major function of the epidermis is to form an effective barrier against water loss and invasion by foreign bodies such as microorganisms. The barrier function is normally conferred by the stratum corneum, the terminally differentiated outer layer of epidermis. Although the epidermis of Fgfr2-IIIb/ mice expresses loricrin, a marker of terminal differentiation, we also tested for acquisition of a functional barrier. As a measure of epidermal barrier function, a vital-dye exclusion assay was performed on foetuses at different stages of development (Fig. 3). Normal mouse embryos become impermeable to the vital dye by E18.5. Despite a severely hypoplastic epidermis, the Fgfr2-IIIb/ mouse embryos established an effective barrier, as judged by dye exclusion. As anticipated, the less affected Fgf10/ mouse embryos also established a barrier. Moreover, the spatial/temporal pattern for establishing the impermeable barrier was maintained (Fig. 3 and data not shown).
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Discussion |
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Dermally produced Fgf10 is a major ligand for epidermal Fgfr2-IIIb, and
mice deficient for Fgf10 also have a hypoplastic epidermis, albeit much less
severe than that seen in the Fgfr2-IIIb/
mice (Ohuchi et al., 2000;
Suzuki et al., 2000
;
Tao et al., 2002
). This
suggests that some other members of the Fgf family may partially compensate
for the loss of Fgf10. The best candidate would be Fgf7, which is also
expressed in the dermis and signals through Fgfr2-IIIb
(Werner et al., 1992
;
Mason et al., 1994
;
Werner et al., 1994
).
Surprisingly, mice deficient for Fgf7 show no developmental skin
abnormalities, nor any significant delay in wound repair, despite a large
increase in Fgf7 transcription that is found in normal skin following wounding
(Werner et al., 1992
;
Werner et al., 1994
;
Guo et al., 1996
). More
recently, Fgf22 has been shown to be expressed in the epidermis and hair
follicles (Nakatake et al.,
2001
; Beyer et al.,
2003
). Its strong structural similarity to Fgf7 and Fgf10 make it
another potential ligand for Fgfr2-IIIb that might act redundantly with Fgf7
and/or Fgf10.
The apparently normal expression of proteins associated with keratinocyte
terminal differentiation is reflected by the attainment of a functional
barrier, as measured by dye exclusion. The barrier starts to form from E16 at
specific epidermal sites, and then spreads around the embryo as a moving front
with the eyelids, ear tips and ventral neck region forming last at E17.5
(Hardman et al., 1998).
Despite the lack of significant epidermal stratification, the barrier function
in Fgfr2-IIIb/ mice is achieved at the same
stage as in wild-type mice (Fig.
3).
Fgfr2-IIIb signalling in hair follicle development
The analysis of hair follicle development showed that mice deficient for
Fgfr2-IIIb exhibit defects both in the number and distribution of hair
follicles (Fig. 6), as well as
producing abnormal hairs (Fig.
7). A reduced number of hair follicles was apparent for both
vibrissal and pelage hair. Interestingly, mice deficient for Fgf10 do not show
a noticeable pelage hair phenotype (Suzuki
et al., 2000), although some disruption of vibrissae similar to in
the Fgfr2-IIIb nulls has been reported
(Ohuchi et al., 2003
).
Following transplantation of Fgfr2-IIIb/
skin onto nude mice, the hairs that grow are abnormally thin and show none of
the patterning of air cells characteristic of normal pelage hairs.
An early marker of hair placode initiation is expression of the
transcription factor Lef1 (Fig.
4). Gene ablation studies have shown that Lef1 is necessary for
the proper development of mammary glands, vibrissae and pelage hairs
(van Genderen et al., 1994).
Mice lacking this gene showed a reduced hair follicle number
(van Genderen et al., 1994
).
However, the Fgfr2-IIIb/ phenotype is not
the same as that found in Lef1/ mice, as the
latter have no vibrissae and the majority of pelage hairs fail to form. This
suggests that Lef1 is more likely to play a role in hair follicle
induction/morphogenesis, whereas Fgfr2-IIIb is more likely to regulate the
numbers of hair placodes induced. Several lines of evidence support this view
(Fig. 8).
|
We propose that these effects are most easily explained by a regulatory
effect of Fgfr2-IIIb signalling on the number of precursor cells needed for
hair follicle formation. Hence, once a follicle placode is initiated the
temporal expression of Lef1, Shh and Bmp4 appear relatively
normal. As hair follicle development in Shh deficient mice appears to
arrest at an early stage, it is possible that Shh acts positively on
Fgf10 expression to maintain follicle growth
(Fig. 8A). This would be
analogous to late limb bud outgrowth, where Shh acts positively to
maintain Fgf10 in the limb bud mesenchyme
(Ohuchi et al., 2000).
The abnormal hair morphology associated with grafts derived from Fgfr2-IIIb/ embryos might result indirectly from the reduced proliferative potential of the epithelial cells, causing too few cells to be available for producing the first wave of hair follicles and resulting in many follicles failing to fully penetrate the subcutis. However, once established, grafted mutant hair follicles show similar levels of proliferation (Fig. 7C,D). If other signalling elements were functioning properly, attempts to drive the cells along the usual differentiation pathway would lead to different types of hair. Instead, only one abnormal hair type was found on grafts from the mutant mice, suggesting that there may also be a direct requirement for Fgf signalling for the patterning and morphology of the different hair types.
Taken together, a common theme appears to be emerging, where the loss of epidermal stratification and compromised hair development derive from a common Fgf-mediated function. We would suggest that proliferation of the ectoderm and the subsequent basal cells is maintained at a sufficient rate to keep pace with the growing embryo, but that the proliferation required to allow the build-up of a multi-layered stratified epithelium is severely restricted. This implies that there may be two pathways regulating cell numbers in the epidermis. One that controls ectoderm and stem cell division on the basement membrane and is Fgf/Fgfr2-IIIb independent, and another that is mediated through Fgf/Fgfr2-IIIb signalling, and that is important for epidermal stratification and normal hair follicle development. The suprabasal cells of the epidermis provide a reservoir to meet the major needs of the developing epidermis, allowing proliferation of basal keratinocytes to be channelled into lateral expansion and the developing hair follicle placodes. The dearth of suprabasal cells in the mutant could require more basal cells to contribute to the maintenance of the epidermis, thereby reducing the number available for lateral expansion (Fig. 8B). This explains the reduction in basal cell density in the mutant and the subsequent reduction in the number of hair follicles.
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ACKNOWLEDGMENTS |
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Footnotes |
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