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First published online 6 December 2006
doi: 10.1242/dev.02721
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1 Department of Cell Biology, Erasmus Medical Center, P.O. Box 1738, 3000 DR
Rotterdam, The Netherlands.
2 Department of Genetics, Erasmus Medical Center, P.O. Box 1738, 3000 DR
Rotterdam, The Netherlands.
3 Department of Obstetrics and Gynecology, Erasmus Medical Center, P.O. Box
1738, 3000 DR Rotterdam, The Netherlands.
* Author for correspondence (e-mail: f.grosveld{at}erasmusmc.nl)
Accepted 31 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Gata3, Hair follicle, Epidermis, Laser capture microscopy, Microarray, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The HF is a mammalian organ that undergoes cyclic
transformations, thereby allowing the study of fundamental developmental
processes (e.g. proliferation, differentiation). The hair shaft and adjacent
multi-layered inner root sheath (IRS) originate from the epithelial matrix
that surrounds the dermal papilla, the permanent mesenchymal portion of the
HF. In response to signals from the papilla, proliferating epithelial cells
protrude into the dermis forming the first HF. Undifferentiated matrix cells
remain in the hair bulb interacting with the dermal papilla
(Cotsarelis et al., 1990;
Oliver and Jahoda, 1988), or
else differentiate as they migrate towards the epidermis.
An outer root sheath (ORS) surrounds the follicle that is contiguous with
and biochemically similar to the basal epidermal layer. The bulge region is
located beneath the sebaceous gland of the HF, marking the lowest point of the
upper part of the follicle that is permanent. It contains a reservoir of stem
cells able to repopulate HF lineages
(Fuchs et al., 2004;
Morris et al., 2004;
Oshima et al., 2001;
Taylor et al., 2000). HFs
undergo self-renewal throughout life. Each hair cycle starts with a growth
phase (anagen) where a germinative layer is formed around the dermal papilla
derived from stem cells present in the bulge
(Morris et al., 2004). Then,
during catagen, the lower epithelial part regresses followed by resting and
shedding periods (the telogen and exogen phases, respectively). Eventually,
the lower part of the follicle grows downwards again to generate a new hair
(Hardy, 1992).
Gata3, a zinc finger transcription factor essential for the proper
development of various tissues and organs
(Pandolfi et al., 1995) is
known to be involved in HF development and skin cell lineage determination
(Ellis et al., 2001;
Kaufman et al., 2003). Using
skin transplantation experiments, Gata3 was shown to be crucially involved in
skin cell lineage determination; its absence resulted in dysfunctional IRS
precursor cells that could not differentiate properly
(Kaufman et al., 2003). To
examine how Gata3 exerts its role in IRS cell lineage determination and to
identify potential Gata3 target genes, we generated
K14-Gata3-/- mice to specifically ablate Gata3
expression, thereby revealing its central role in mouse epidermis and HF
development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Laser-capture microdissection and microarray analysis
Laser-capture microdissection was performed on 10 µm cryosections using
the PALM MicroBeam microscope system (PALM Microlaser Technologies). RNA was
analysed as described
(http://www.affymetrix.com/index.affx).
Microarray chips were analyzed with Affymetrix GeneChip software. Microarray
data complied with the MIAME regulations and are available in ArrayExpress
(accession code: 5988).
In situ hybridization, TUNEL and skin barrier
In situ hybridization with antisense digoxigenin-UTP-labelled RNA probes on
10 µm sections of skin samples was performed as described
(Schaeren-Wiemers and Gerfin-Moser,
1993). Cell death was detected with the Cell Death Detection Kit,
Fluorescein (Roche). Skin barrier experiments were performed as described
(Hardman et al., 1998).
Immunohistochemistry, X-Gal staining and BrdU labelling
Mice were injected with 50 mg/kg bodyweight BrdU and sacrificed 2 hours
later. Cryosections were fixed for 10 minutes in 4% PFA in PBS, then blocked
with 1% BSA, 0.05% Tween in PBS. Primary antibodies were: K6 (rabbit, 1:5,000;
Covance, PRB-169P), K14 (rabbit, 1:10,000; Covance, PRB-155P), Gata3 (mouse,
1:200; Santa Cruz, HCG3-31), CD3 (rat, 1:100; Santa Cruz, KT3), cyclin A2
(rabbit, 1:100; Santa Cruz, C-19), Cdk4 (rabbit, 1:100; Santa Cruz, C-22),
cyclin E1 (rabbit, 1:100; Santa Cruz, M-20), loricrin (rabbit, 1:500; Covance,
PRB 145P), K14 (rabbit, 1:10,000: Covance, PRB_155P), ß-catenin (mouse,
1:100; BD, #14), AE13 (mouse, 1:20) (Lynch
et al., 1986), AE15 (mouse, 1:10)
(O'Guin et al., 1992), MTS24
(rat, 1:200) (Gill et al.,
2002), BrdU (mouse, 1:100; DAKO, #Bu20a) and K10 (mouse, 1:50;
Sigma, #k8.60). Relevant FITC-, TxR- or HRP-conjugated goat secondary
antibodies (1:100, DAKO) were used. For BrdU immunohistochemistry, tissue was
fixed in 4% PFA, 4°C overnight and subsequently embedded in paraffin and
sectioned at 7 µm. After deparaffination, sections were boiled in 0.01 M
citrate buffer (pH 6.0) for 15 minutes prior to primary antibody incubation.
For X-Gal staining, sections were fixed for 1 minute in 0.5% glutaraldehyde,
1% PFA, and incubated in X-Gal staining solution for 5 hours at room
temperature. Images were taken with an Olympus BX40 microscope and Axio Imager
(Zeiss) fluorescence microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Vassar et al., 1991). These
mice were crossed with conditional Gata3 knockout (Gata3cko)
mice (Fig. 1A). The human K14
promoter drives Cre expression (visualized in a floxed Rosa-LacZ
reporter mouse line) in epidermis, all HF cell types, salivary gland, mammary
epithelium and epithelial cells from other tissues
(Jonkers et al., 2001). We
detected Cre activity in the epidermis at embryonic day (E) 13.5,
whereas placodes were Cre-positive two days before the onset of
Gata3 expression (i.e. E15.5). Heterozygous skin-specific
Gata3 knockouts were indistinguishable from wild-type (wt)
littermates (not shown). K14cre/Gata3cko/wt mice were crossed with
heterozygous and homozygous Gata3cko mice to generate
K14cre/Gata3cko mice (referred to as K14-Gata3-/-
or ko mice). These lacked expression of Gata3 in the hair follicles and
epidermis at all stages, as determined by immunohistochemistry (not
shown).
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Delayed hair growth and disturbed hair maintenance in K14-Gata3-/- mice
In contrast to wt, where external hairs were visible from P7, forming a
well-developed fur coat by about P10, K14-Gata3-/- mice
remained completely bald (Fig.
1B). From P12 onwards, hair growth was sparse around the head and
neck resulting in a thin fur coat with short and stubby hairs (P17,
Fig. 1C). The delayed onset of
hair growth was followed by the gradual loss of abdominal hair (P19,
Fig. 1J,K) and an anterior to
posterior hair shedding in the head area
(Fig. 1L,M), with limited hair
regeneration. Remaining hairs appeared irregular, with a short, thick and hard
appearance and a thick-rounded tip instead of the normally observed thin tip
(Fig. 1N-R). Whiskers appeared
short and thick (Fig.
1E,F,S,T).
Aberrant hair follicle organization and abnormal hair pigmentation in K14-Gata3-/- mice
At P0, K14-Gata3-/- HFs appeared normal. However,
beginning at P3, K14-Gata3-/- HFs were abnormal and grew
at a much wider angle, often parallel to the skin surface, never reaching the
epidermis (Fig. 2H). However,
the overall number of HFs was not reduced, suggesting that Gata3 is not
essential during HF induction. Whereas wt hairs had the typical ladder-like
appearance resulting from the large, keratinized pigment-containing cells
interspersed with air pockets in the hair medulla, the
K14-Gata3-/- hair pigment deposition was disorganized and
without air spaces (Fig. 1R,
Fig. 2F) and the sebaceous
glands were enlarged from P7 onwards. Subsequently, the
K14-Gata3-/- HF entered telogen after a delayed catagen
lasting at least 6 days instead of the normal 3 days (see Fig. S1A-F in the
supplementary material) and the HF proximal end failed to reform (see Fig.
S1I,J in the supplementary material). New visible hairs were not produced,
although some developing HFs and small cysts were visible (see Fig. S1J in the
supplementary material).
The newly formed HFs that derive from stem cells
(Morris et al., 2004) are
known to migrate from the bulge to the matrix area of the hair bulb, thereby
forming a germinative layer that becomes committed to distinct HF cell
lineages (Legue and Nicolas,
2005). The lack of HF renewal prompted us to investigate whether
Gata3 is also expressed in cells other than those of the IRS
(Kaufman et al., 2003).
Evaluation of Gata3 expression in Gata3 LacZ knock-in mice and by in situ
hybridization revealed Gata3 to be readily expressed in other skin structures
(Pata et al., 1999)
(Fig. 3 and see Fig. S2 in the
supplementary material). At E15.5, Gata3 was expressed in the
epidermis (Fig. 3A), whereas
during anagen Gata3 was also expressed in the IRS, ORS, sebaceous
glands, epidermis and infundibulum (Fig.
3B-L, and see Fig. S2 in the supplementary material) and in
certain germinative layer cells surrounding the dermal papilla
(Fig. 3F,J,L). During catagen,
Gata3 expression was restricted to the IRS
(Fig. 3G), whereas during
telogen it was present in the epidermis and sebaceous glands, butabsent from
the HF (Fig. 3H).
Epidermal hyperplasia and hyperkeratosis in K14-Gata3-/- skin
Instead of developing new HF bulbs, we noticed pronounced hyperplasia in
the K14-Gata3-/- epidermis (see below). Despite a slightly
delayed barrier function in fetuses at E16.5, E17.5 and E18.5 (data not
shown), initial development of the epidermis appeared normal in skin sections
from newborn mice (P0, Fig. 2A,B and
P3, Fig. 2C,D). At
P3, skin hyperplasia was followed by wrinkles
(Fig. 2E-H) covered with
squames (Fig. 1T).
K14-Gata3-/- skin sections, at different ages, revealed
gradual, pronounced epidermal thickening including that of basal and
suprabasal layers (see Fig. S3B,D in the supplementary material) as well as
the development of hyperkeratosis (increased thickness of stratum corneum;
Fig. 2C-H and see Fig. S3B,C in
the supplementary material). We did not observe any nuclei in the squames of
stratum corneum that would mark the presence of immaturely shed keratinocytes,
a feature that is routinely observed in psoriasis. In contrast to the
thickened epidermis, the dermis of K14-Gata3-/- pups
appeared substantially thinner, along with a reduction in subcutaneous adipose
tissue as compared with wt littermates
(Fig. 2G,H).
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80 genes expected
to occur by chance under these selection criteria.
To avoid any preselection of genes and thus the potential introduction of
bias, we used a previously described methodology
(Hosack et al., 2003) to
identify all over-represented biological processes in our dataset [i.e. cell
cycle, epithelial growth and differentiation, immune and defense responses,
signal transduction pathways and energy metabolism
(Fig. 4G)]. Analysis of these
processes (Fig. 4H, and see
Table S1 in the supplementary material) led us to identify:
PCR evaluation of the expression levels of several genes in the HF of K14-Gata3-/- mice and wt littermates confirmed the validity of the microarray data (see Fig. S7 in the supplementary material).
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Keratins and keratin-associated proteins in K14-Gata3-/- mouse skin
Of the 34 keratin and keratin-associated proteins whose expression was
significantly altered, 30 were overexpressed in
K14-Gata3-/- mice. In particular, we noticed the increased
expression of the Krtap16 gene family, ranging from 6- to 33-fold, which
was further confirmed by in situ mRNA evaluation of Krtap16-7
(Fig. 6A,B). Its expression
domain was not separate from the pigmented cells, suggesting that the cortex
and medulla are no longer separated (Fig.
6A,B). Consistent with previous findings, AE13 antibody detected
the acidic hair keratins expressed in the cortex and cuticle of the hair shaft
(Lynch et al., 1986)
(Fig. 6C), whereas
K14-Gata3-/- HFs exhibited expanded precortex and cortex
when compared with wt HFs (Kaufman et al.,
2003) (Fig. 6D).
Similar to Krtap16-7, the expression domain detected with the AE13
antibody was not separate from the pigmented cells
(Fig. 6D, arrowheads).
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) (Fig.
6E), detected a thin bundle of AE15(+)
K14-Gata3-/- IRS cells
(Kaufman et al., 2003)
(Fig. 6F). Double staining with
AE15 and K14 antibodies revealed that the companion layer did not separate
AE15(+) and K14(+) cells (Fig.
6E,F).
Consistent with the absence of Huxley's layer and the cuticle of the IRS in
K14-Gata3-/- HFs, expression of µ-crystallin
(Crym - Mouse Genome Informatics), known to co-localize with Gata3 in
the IRS (Fig. 6G)
(Aoki et al., 2000), was
diminished in K14-Gata3-/- HFs
(Fig. 4H and
Fig. 6H).
Next, we examined the differentiation status of
K14-Gata3-/- mouse skin. Staining of loricrin, a
late-stage differentiation marker, showed no differences in both young (P1,
Fig. 7A,B) and mature (5
months, Fig. 7C,D) skin. In P4
skin, K10 expression was not different between the wt and
K14-Gata3-/- mice (Fig.
7E,F). In P26, K10 was expressed in a layer surrounding the
proximal part of K14-Gata3-/- HF, the ORS and the basal
epidermal keratinocytes (Fig.
7H). In wt skin, however, it was restricted to suprabasal
epidermal keratinocytes and infundibulum
(Fig. 7G). K6, a cytokeratin
known to mark proliferating cells proximal to the bulge and K10, was observed
distal to the bulge and in the basal epidermis
(Fig. 7I-K). Occasionally, K10
and K6 colocalized (Fig. 7K).
At later stages, the number of K6-expressing cells in the skin increased
substantially (Fig. 7L,M).
Immunohistochemical analysis of K14, typically expressed in the ORS and the
basal layer of stratified squamous epithelia of normal HFs
(Vassar et al., 1989),
confirmed our findings (Fig.
4H). Interestingly, the basal epidermal layer and ORS were almost
twice as thick in K14-Gata3-/- compared with wt skin
(Fig. 6E,F). Together, these
changes indicate aberrant differentiation, which is likely to be due to the
deregulation of signal transduction pathways.
Signal transduction and embryonic development in K14-Gata3-/- HFs
Most of the prominent signal transduction pathways known to be involved in
HF morphogenesis were significantly over-represented in the
K14-Gata3-/- HF transcriptome, including the Wnt and BMP
families, sonic hedgehog and Notch (Fig.
4G-H). Wnt3, Wnt10b, and Wnt11 were upregulated
in K14-Gata3-/- HFs, whereas Wnt5a and
Wnt6 were downregulated compared with controls
(Fig. 4H). In the wt HF,
Wnt5a expression was detected in the ORS, IRS and dermal papilla
(Fig. 8A), but was almost
undetectable in K14-Gata3-/- HFs
(Fig. 4H and
Fig. 8B), a finding in
agreement with the absence of differentiated IRS cells, the significant
decrease in Shh mRNA levels (see below) and the known absence of
Wnt5a expression in Shh-/- mice
(Reddy et al., 2001).
Wnt11 was previously shown to be expressed in the outermost layers of
the HF, ORS and dermal sheath, above the dermal papilla
(Reddy et al., 2001)
(Fig. 8C). Similar to K10
(Fig. 7H), Wnt11
expression was expanded to the lowest (most proximal) part of the bulb in
K14-Gata3-/- HFs (Fig.
8D), and was also present in the K14-Gata3-/-
outer HF layer, as compared with controls. This could originate from an
increased number of precursor IRS cells in the matrix that are in turn likely
to be due to a migration defect that could also underlie the observed
thickening of the matrix. Frizzled Wnt receptors Fzd1, Fzd3 and
Fzd6 were overexpressed in the K14-Gata3-/- HF,
whereas frizzled-related protein (Frzb, an inhibitor of Wnt
signaling) was significantly downregulated
(Fig. 4H). In agreement, we
detected strong nuclear ß-catenin staining in
K14-Gata3-/- HFs (Fig.
8E,F). Shh along with Gli1, which is a known
target and transducer of Shh signaling, the hedgehoginteracting protein
(Hhip) and patched homolog 2 (Ptch2) demonstrated reduced
expression levels in K14-Gata3-/- HFs
(Fig. 4H). The decrease in
Gli1 expression levels was also confirmed by in situ hybridization in
P7 K14-Gata3-/- HFs
(Fig. 8G,H), particularly in
the region of the dermal papilla.
Whereas Notch1 was upregulated in the
K14-Gata3-/- HF (Fig.
4H), the expression levels of Notch2 and Notch3
were not significantly affected. In situ mRNA hybridization showed
Notch1 to be expressed in precursor cells of cortex, cuticle, and IRS
(Fig. 8I)
(Kopan and Weintraub, 1993).
Although the expression pattern of Notch1 appeared unchanged in the
K14-Gata3-/- HF, its expression levels were substantially
higher, particularly in the lower matrix cells
(Fig. 8J). Taken together,
these findings support the notion that Gata3 plays a central role in Shh and
Notch signaling pathways, though this effect is likely to be indirect as
Shh and Notch are expressed in different cells.
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).
Cutl1, which encodes the transcriptional repressor CDP known to be
involved in epidermal differentiation, was downregulated in the
K14-Gata3-/- mice (Fig.
4H). However, the hair phenotype of Cutl1-/-
mice (e.g. circle hairs, corkscrew hairs) as well as several documented
transcriptional changes are different to those of
K14-Gata3-/- mice
(Ellis et al., 2001),
suggesting that CDP and Gata3 are likely to function in distinct signaling
pathways.
Stem cell markers in K14-Gata3-/- skin
To analyze the presence and distribution of bulge cells in the
K14-Gata3-/- skin and to delineate the role of Gata3 in
the formation of the bulge, we investigated the expression of stem cell
markers keratin 15 and S100A6 (Blanpain et
al., 2004; Kizawa and Ito,
2005; Cotsarelis,
2006). Compared to wt skin, keratin 15 and S100A6 were
dramatically upregulated in the distal K14-Gata3-/- HF and
were also present in the epidermis at P11, P19 and 5 months. Whereas the HF
and epidermis expression domains are disconnected in wt, they form a continuum
in the K14-Gata3-/- mice (see Fig. S6 in the supplementary
material), suggesting that the bulge contributes more to the knockout
epidermis and that Gata3 function is different in the epidermis than in the
HF. As a result, the bulge also appears to contribute to epidermal thickening
in addition to the increased proliferation of the epidermal cells
(Fig. 5A,B). To obtain
additional evidence for the contribution of the HF to the epidermis, we
examined the bald inside of the ear versus the outside, which does have many
HF. Interestingly, K6, the marker for proliferating cells, is absent in
epidermis from both the inside and outside of the ear, but is present in HF
(Fig. 9A). In
K14-Gata3-/- the epidermis on the outside of the ear
expresses K6, whereas the inside, that never had HFs, remains K6-negative
(Fig. 9B).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the
present study, Gata3 is also expressed in layers other than the IRS of the HF,
and all layers are disturbed. Normally, cells in the bulge reform the proximal
part of the HF to replace shed hairs by new ones. This process does not take
place in the K14-Gata3-/- knockout resulting in bald mice.
Despite their disoriented appearance, the number of HFs was not substantially
reduced, suggesting that Gata3 is not essential for the early steps of HF
induction. Instead, Gata3 appears to have a role in hair cycling, as bulge
cells no longer give rise to a new follicle. This is consistent with
Gata3-/- skin grafting experiments showing that, in the
absence of Gata3, the hair structures are still apparent in very early skin
although with an aberrant morphology. The Gata3-/- skin
was not analyzed at later stages in those transplantation experiments
(Kaufman et al., 2003).
Gata3 impinges on the distribution of proliferating cells in the hair follicle matrix
Despite significant advances in our understanding of the role of Gata3 in
HF development, little is known about those biological processes most
significantly affected in the absence of Gata3 in the murine epidermis and
HFs. We implemented a full mouse transcriptome analysis in laser-captured HFs
to get an unbiased insight into: (1) the implicated biological processes; (2)
the signalling mechanisms involved; and (3) the nature of the defect itself.
This approach revealed several processes most pertinent to the cell cycle,
epithelial growth and differentiation and signal transduction pathways.
Importantly, we identified a broad, uniform decrease in the expression of most
genes associated with the transition of the mitotic cell cycle. However,
subsequent evaluation with BrdU staining revealed that, although the overall
number of BrdU(+) cells did not differ between the
K14-Gata3-/- and wt HFs, there was a significant
difference in the distribution of proliferating cells throughout the HF. These
findings highlight the direct role of Gata3 in differentially regulating
specific cell lineages that originate from the matrix area and, together with
previous data (Kaufman et al.,
2003), suggest that loss of Gata3 negatively impacts on
proliferation and cell fate decisions in the matrix, which is likely to shift
matrix cells towards fates other than the inner root sheath. Consistent with
the decreased proliferative capacity of those cells located in the basal layer
of the HF, apoptosis was reduced in the K14-Gata3-/- HF,
which is likely to reflect the delayed onset of growth and differentiation. In
agreement, several pro-apoptotic genes were downregulated whereas
anti-apoptotic genes were upregulated. Coupled to this response, a number of
genes involved in histone metabolism and chromatin modification were
significantly upregulated in the absence of Gata3 from the HF.
Epithelial growth and differentiation represent significantly affected processes in the K14-Gata3-/- HF
Notably, changes in expression of genes encoding keratins and
keratin-associated proteins were amongst the broadest identified. IRS cells
express a number of genes that are also expressed in the suprabasal epidermal
layers, suggesting that both cell types employ comparable differentiation
pathways (Botchkarev and Paus,
2003). K10 expression is activated in terminally differentiated
epidermal keratinocytes, when they start losing their proliferative
competence. Conversely, K10 expression is severely reduced under conditions
that promote proliferation (Fuchs and
Green, 1980; Fuchs and Weber,
1994; Moll et al.,
1982), whereas ectopic K10 expression can induce an Rb-mediated
cell cycle arrest (Paramio et al.,
1999; Santos et al.,
2002). By contrast, K14-Gata3-/- mice express
(ectopic) K10 in proliferating basal epidermal cells and in the decreased
proliferating cells in the bulb of the HF. By analogy to Gata1
(Rodriguez et al., 2005), this
suggests that Gata3 has (at least) a dual regulatory function affecting a
number of genes involved in cell proliferation and terminal
differentiation.
K10 expression is also regulated by the transcription factors C/EBP and
AP-2 (Tcfap2a - Mouse Genome Informatics)
(Maytin et al., 1999). Despite
certain differences between the Cebpß-/- and
K14-Gata3-/- mice, some phenotypic parallels are striking.
Although in Cebpß-/- mice the epidermis, the dermis
and the size and number of HFs appear normal, K10 expression is expanded
similar to the K14-Gata3-/- mice and subcutaneous fat is
also decreased (Maytin et al.,
1999). Interestingly, the two C/EBP-binding sites in the
K10 (Krt10 - Mouse Genome Informatics) promoter flank a
highly conserved GATA-binding site, suggesting that Gata3 and C/EBP regulate
K10.
Wnt and BMP signalling pathways in the K14-Gata3-/- hair follicle
Wnt signaling regulates HF development. Mice lacking Lef1, a downstream Wnt
mediator, demonstrate a reduced number of body hairs
(van Genderen et al., 1994),
whereas ectopic expression of either Lef1 or constitutively active
ß-catenin induces ectopic HFs (Gat et
al., 1998; Noramly et al.,
1999; Zhou et al.,
1995). Upon Wnt signaling, ß-catenin accumulates in the
cytoplasm and is transported to the nucleus, where it interacts with members
of the LEF/TCF family of transcription factors, thereby activating the
expression of downstream gene targets
(Barker et al., 2000).
K14-Gata3-/- show a considerable change in Wnt-related
protein expression (Fig. 4,
Fig. 8E,F), which probably
underlies the abnormal hair formation
(DasGupta and Fuchs, 1999).
Our results are in agreement with those obtained in
Bmpr1a-/- mice, which have decreased levels of Gata3
(Andl et al., 2004;
Ming Kwan, 2004;
Yuhki et al., 2004).
Interestingly, all BMP family genes whose expression changes significantly
in the K14-Gata3-/- mice (Bmp6, Bmp8a and
Bmp15) were upregulated (Fig.
5H). Moderately elevated Bmp6 signalling leads to increased
proliferation of basal epidermal keratinocytes
(Blessing et al., 1996;
Botchkarev et al., 1999),
similar to that observed in the K14-Gata3-/- basal
epidermal layer. However, the phenotype of mice overexpressing Bmp6 in the
suprabasal epidermis (Blessing et al.,
1996) is distinct from that seen in
K14-Gata3-/- mice.
FGF and Notch1 signalling pathways in the K14-Gata3-/- hair follicle
Of all FGF family members (see Table S1 in the supplementary material) only
Fgf5 was downregulated in K14-Gata3-/- mice,
whereas Fgfr2 demonstrated increased mRNA expression levels. Mutation
of Fgf5 (Hebert et al.,
1994) or expression of a dominant negative Fgfr2
(Schlake, 2005) leads to the
growth of long thin hair, whereas Fgfr2-/- mice
(Petiot et al., 2003) have a
decreased number of HFs, impaired hair formation and reduced basal epidermal
cell proliferation. Although, the number of HFs was not affected in
K14-Gata3-/- mice, Fgf5 was downregulated, as
opposed to increased expression of its receptor, suggesting that the receptor
expression is likely to be rate-limiting. Lastly, Notch1 was upregulated in
K14-Gata3-/- mice. As Notch1 expression, driven by
involucrin in the IRS and suprabasal epidermis
(Uyttendaele et al., 2004),
provokes a similar, yet less severe hair phenotype, our findings highlight the
possibility of Notch1 directly affecting hair formation.
In essence, several transgenic and/or knockout mouse models display
phenotypic overlap (e.g. epidermal hyperplasia, reduced HF growth). In the
majority of these cases, the hair phenotype is frequently accompanied by
changes in adipogenesis and fat metabolism, previously thought to be an
indirect effect. In addition, increased or reduced expression levels for
certain genes, including Hoxc13
(Godwin and Capecchi, 1998;
Tkatchenko et al., 2001), BMP
genes (Blessing et al., 1996;
Botchkarev et al., 2002) and
Notch1 (Uyttendaele et al.,
2004; Vauclair et al.,
2005), associated with the signaling mechanisms in hair formation
appear to exert similar effects. However, for the majority of genes, it
becomes increasingly apparent that their exact temporal and spatial expression
is crucial; whereas reduction in Shh expression levels results in
disturbed HF growth (St-Jacques et al.,
1998), its increase leads to epidermal hyper-proliferation with
distinct phenotypic features (Ellis et
al., 2003).
Our results show that Gata3 is a key transcription factor that impinges on
the regulation of several processes resulting in a composite of the phenotypes
described above. The changes in keratin expression suggest that Gata3 acts as
a moderator between HF development and epidermal differentiation through the
orchestrated regulation of distinct signal transduction pathways. If Gata3
levels are greatly diminished, the epidermis hyperproliferates and HF matrix
cells do not develop into functional IRS cells. Instead, the HF appears to
contribute to the basal epidermis. This switch in mode of action has
previously been proposed for Notch1
(Uyttendaele et al., 2004), a
negatively regulated target of Gata3. In turn, BMPs regulate Gata3
(Andl et al., 2004;
Kobielak et al., 2003) and
vice versa (this paper) to maintain appropriate Gata3 expression levels. We
therefore propose that Gata3 is a crucial component in the choice between
forming different layers of the HF versus basal epidermal cells maintaining
their balance through the coordinated regulation of the BMP and Notch
signaling pathways.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/2/261/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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0.05; Student's
t-test. Scale bar in A: 150 µm for A,B,D,E,J,K; 100 µm for
G,H.
