1 Institut de Génétique et de Biologie Moléculaire et
Cellulaire (IGBMC), CNRS, INSERM, ULP, BP 10142-67404, Illkirch, C.U. de
Strasbourg, France
2 Institut Clinique de la Souris (ICS), BP 10142, CU de Strasbourg, 67404
Illkirch, France
3 Collège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05,
France
4 Unité Expression Génétique et Maladies, CNRS URA 1644,
Département de Biologie du Développement, Institut Pasteur,
75724 Paris, France
* Authors for correspondence (e-mail: metzger{at}igbmc.u-strasbg.fr and chambon{at}igbmc.u-strasbg.fr)
Accepted 1 August 2005
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SUMMARY |
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Key words: Targeted somatic mutagenesis, Cre-Lox, Epidermis, Limb, SNF2ß-BRG1
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Introduction |
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As mice lacking BRM are viable and fertile
(Reyes et al., 1998), it was
suggested that BRM and BRG1 might be functionally redundant. However, in vitro
studies indicated that the two factors might each be involved in regulating
the expression of different sets of genes
(Kadam and Emerson, 2003
).
Moreover, Brg1 ablation in murine F9 embryonal carcinoma cells
results in cell death, thus demonstrating that BRG1 plays crucial,
non-redundant functions (Sumi-Ichinose et
al., 1997
). However, it is not a general cell viability factor, as
Brg1-null mouse fibroblasts are viable
(Bultman et al., 2000
).
Lethality of Brg1-null mouse embryos during the peri-implantation
stage demonstrated that BRG1 exerts specific functions early in development,
but precluded the elucidation of its function(s) at later stages in mammals
(Bultman et al., 2000
).
Interestingly, Cre-mediated ablation of Brg1 during T cell
development revealed essential roles of this factor at various stages of T
cell differentiation (Chi et al.,
2003
).
The skin, which consists of the epidermis and underlying dermis, is a very
attractive tissue in which to study the in vivo functions of genes that
regulate the expression of proteins involved in the control of cellular
proliferation and differentiation. During embryonic development, the
ectodermal cell layer covering the body develops into a stratified epidermis
that is essential at birth, when the organism confronts the arid and toxic
postnatal environments. The first sign of stratification of mouse embryonic
epidermis occurs at embryonic day (E) 9.5, when the periderm forms. Epidermal
maturation continues with the formation of the future spinous layer at E15.5,
and by E18.5 the epidermis is fully differentiated
(Byrne et al., 2003).
Keratinocytes from the basal layer periodically withdraw from the cell cycle
and commit to terminal differentiation, while migrating to the next layers.
The outermost layer of the skin (statum corneum) is composed of mechanically
tough, dead, cornified cells (squames), which develop as a result of a complex
terminal differentiation program, and provide vital physical and permeability
barriers to vertebrates (Kalinin et al.,
2002
). Formation of the epidermal permeability barrier requires
the delivery of lipids and proteins, which are contained in lamellar granules
(keratinosomes) present in keratinocytes of the granular layer, to the stratum
corneum interstices, as well as the formation of high molecular weight
polymers through the crosslinking of corneocyte envelope proteins (loricrin,
involucrin, filagrin and other peptides) and packing of corneocytes by
corneodesmosomes. Epidermal homeostasis relies on a tightly regulated balance
between keratinocyte proliferation and differentiation, the alteration of this
leads to various skin diseases (Watt,
2000
).
To investigate the role of BRG1 in skin ontogenesis, we established mice
bearing LoxP-flanked (floxed) Brg1 alleles and ablated Brg1
in the forming epidermis using K14-Cre (Li
et al., 2001) or K14-Cre-ERT2
(Indra et al., 2000
;
Li et al., 2000
) transgenic
mice that express either the bacteriophage P1 Cre-recombinase or the
ligand-dependent Cre-ERT2 recombinase under the control of the
human K14 promoter, which is active in the surface ectoderm and the basal
layer of the epidermis (Vassar et al.,
1989
). We show that BRG1 is dispensable for embryonic epidermis
formation, but is essential for establishing the skin barrier at later fetal
stages. Ablation of Brg1 in the forming epidermis before E12.5 also
induces developmental limb defects that are by-passed by temporally controlled
Brg1 ablation at later times. Moreover, ablation of Brg1 in
epidermal keratinocytes of mice lacking BRM revealed that the SWI2/SNF2 ATPase
subunits of the chromatin remodelling complexes are not essential for
keratinocyte proliferation and `early' differentiation, and that BRM can
partially substitute for BRG1 function during keratinocyte `late' terminal
differentiation.
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Materials and methods |
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Tamoxifen administration
Tam (0.1 mg in 50 µl sunflower oil), prepared as described
(Metzger et al., 2003), and
oil (vehicle) were injected intraperitonially to pregnant females.
Genotyping of Brg1 alleles
Genomic DNA was isolated from fetal skin and adult tail skin, and, whenever
required, the dermis and epidermis was separated
(Li et al., 2001). The various
Brg1 alleles were identified by PCR with P1, P2 and P3 primers: (+)
allele, P1-P2 (241 bp); (L2) allele, P1-P2 (387 bp); (L-) allele, P3-P2 (313
bp) (Sumi-Ichinose et al.,
1997
).
X-Gal staining, in situ RNA analysis and skeletal analysis
For whole-mount X-Gal staining, embryos recovered between E9.5 and E10.5
were fixed in 2% formaldehyde, rinsed in PBS (pH 7.4) and incubated in
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) solution in PBS
overnight at 37°C in the dark (Byrne et
al., 1994), washed twice in PBS and photographed. X-Gal staining
of 10 µm thick frozen sections was performed as described
(Indra et al., 1999
).
Whole-mount Fgf8 in situ hybridization was as described
(Wendling et al., 2001
).
Skeletons of E18.5 fetuses were prepared and stained as described
(Lufkin et al., 1992
).
Semithin and electron microscopic analysis
Limb buds were collected from E11.5 foetuses. Skin biopsies were matched
for age and body sites. Semithin (2 µm section) and transmission electron
microscopy samples were processed as described
(Li et al., 2001;
Segre et al., 1999
). For
scanning electron microscopy (SEM), samples were prepared as described
(Choi et al., 1997
).
Immunohistochemistry
After fixation in 2% paraformaldehyde (PFA), 10 µm skin or limb
cryosections were blocked in 5% normal goat serum (NGS, Vector), incubated
overnight with a polyclonal rabbit anti-BRG1 antibody (1:1000) at 4°C
(Sumi-Ichinose et al., 1997),
washed in PBS/0.1% Tween 20, and incubated for 1 hour at room temperature with
a CY3-conjugated donkey anti-rabbit antibody (1:400) (Jackson ImmunoResearch)
or an Alexa-conjugated goat anti-rabbit antibody (1:200) (Interchim, France).
BRM and KI-67 immunohistochemistry was performed on 10 µm dorsal skin
cryosections as decribed (Reyes et al.,
1998
; Li et al.,
2001
). Counterstaining was performed with DAPI.
Skin permeability assay
X-Gal permeability was performed according to Hardman et al.
(Hardman et al., 1998).
Briefly, freshly isolated E18.5 fetuses were rinsed in PBS, immersed in X-Gal
solution, pH 4.5, at 37°C for 8 hours, washed in PBS for 1-2 minutes and
photographed.
In vivo transdermal absorption of the fluorescent dye Lucifer yellow
E18.5 fetuses were restrained in Petri dishes with their backs in contact
with 1 mM Lucifer Yellow in PBS (pH 7.4) at 37°C, as described
(Matsuki et al., 1998). After
a 1-hour incubation, fetuses were sacrificed, frozen and cryosectioned
dorsoventrally at a thickness of 5 µm. DAPI counterstained sections were
analysed by fluorescence microscopy.
Trans-epidermal water loss (TEWL) measurement
Dorsal and ventral skin TEWL was determined on E18.5 fetuses with a Cortex
Technology's Dermalab system (Denmark), equipped with a TEWL probe. Mean
values of six measurements per animal were determined. Data are expressed in
g/h/m2, as means±s.e.m. from four animals.
Quantitative RT-PCR
Total RNA was extracted from approximately 100 mg skin of E18.5 fetuses
using Trizol® Reagent (GIBCO BRL Life technologies). cDNA was synthesised
from 5 µg RNA using the SuperscriptTM II kit (Invitrogen) according to
the manufacturer's instructions. Real-time PCR was performed with a
LightCycler (Roche Diagnostics) and the SYBR green kit. For that purpose, 5
µl of cDNA was diluted to 20 µl, and 2 µl of the diluted cDNA was
used for each amplification. Amplification specificity was verified by
melting-curve analysis and the data quantified with the LightCycler software.
Alternatively amplified products from the exponential phase (between 20-25
cycles) were electrophoresed on 2% agarose gel, transferred onto nylon
membrane and probed with radiolabelled oligonucleotides. Primers used were as
follows.
The radiolabeled oligonucleotide probes were:
Statistical analyses
Where relevant, data were compared by Unpaired Student's t-test,
with corrections for unequal variance made using the Statview 5 programme
(Abacus Concepts, CA, USA). Values are reported as mean±s.e.m. and the
significance level was set at P=0.05.
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Results |
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Efficient Brg1 ablation in the limb ectoderm before E11.5 and in epidermal keratinocytes of K14-Cretg/0/Brg1L2/L2 fetuses during skin morphogenesis
We have previously shown that the Cre recombinase expressed under the
control of the human K14 promoter in K14-Cre transgenic mice
efficiently mediates site-specific recombination at loxP sites in
keratinocytes of fetal and adult epidermis
(Li et al., 2001). To further
characterise Cre activity during skin ontogenesis, hemizygote
K14-Cretg/0 mice were bred with RosaR26R reporter
mice (called thereafter ROSAfl/+) that express
ß-galactosidase after Cre-mediated recombination
(Soriano, 1999
). In E9.5
K14-Cretg/0/ROSAfl/+ bigenic fetuses,
X-Gal staining was patchy, but mainly located in the caudal region and forming
forelimb bud (Fig. 1C, compare
parts a and b). By E10, it was still restricted to the posterior region of the
embryo and limb buds (Fig. 1C,
part c, and data not shown). Note that staining of the `forming' hindlimb bud
appeared stronger than that of the forelimb bud. Histological analyses
revealed that Cre-mediated excision was restricted to the ectodermal layer
(data not shown). At E12.5, X-Gal staining was uniformly distributed in the
surface ectoderm, including that of limbs, and after E14.5 most, if not all,
epidermal keratinocytes were X-Gal stained
(Fig. 1D, and data not
shown).
Immunohistochemical analysis of E10.5, E11.0 and E12.5 control embryos revealed that BRG1 was uniformly expressed in the surface ectoderm, including that of outgrowing limbs (Fig. 2A,B, and data not shown). At E18.5, it was strongly expressed in most, if not all, basal cells, as well as in about 70% of the spinous and 30% of the granular cells (Fig. 2C, parts a and b, and data not shown). To ablate Brg1 in the forming epidermis, Brg1L2/L2 mice were bred with K14-Cretg/0 mice, to generate K14-Cretg/0/Brg1L2/L2 fetuses. L-, but no L2, alleles were detected in the epidermis of E18.5 K14-Cretg/0/Brg1L2/L2 fetuses, whereas only L2 alleles were present in their dermis (see Fig. 2D, lanes 1 and 2), thus demonstrating that Brg1 was efficiently and selectively ablated in keratinocytes of the forming epidermis. In agreement with these results, no BRG1 protein was revealed by immunohistochemistry in epidermal keratinocytes of E18.5 K14-Cretg/0/Brg1L2/L2 fetuses [hereafter called Brg1ep-/-(c) mice] (Fig. 2C, parts c and d). Note that Brm transcript levels were similar in the skin of E18.5 control and Brg1ep-/-(c) fetuses (data not shown). At E10.5, the BRG1 protein expression pattern was similar in the surface ectoderm of Brg1L2/L2 (control) and Brg1ep-/-(c) fetuses, but, at E11, BRG1 was decreased in the cells of the surface ectoderm of Brg1ep-/-(c) forelimbs and was very low in mutant hindlimbs (Fig. 2A; and data not shown). At E11.5, BRG1 protein was not detected in either fore- or hindlimb ectoderm (data not shown). Moreover, BRG1 protein was observed in less than 50% of the cells of the dorsal ectoderm of E11.5 Brg1ep-/-(c) fetuses, and, at E12.5, almost all ectodermal cells were BRG1-depleted (Fig. 2B; and data not shown). Thus, Brg1 is efficiently ablated in the limb ectoderm at E11.5, and in the developing epidermis of Brg1ep-/-(c) fetuses before E12.5.
Brg1 ablation in the limb ectoderm of Brg1ep-/-(c) fetuses induces severe hindlimb defects
E18.5 Brg1ep-/-(c) mutant fetuses were obtained at the
expected mendelian ratio. Their forelimbs were properly developed, but their
hindlimbs and tails were malformed (Fig.
3A, and data not shown). The severity of the hindlimb defects was
variable, ranging from five abnormal digits and a normal zeugopod (tibia and
fibula), to a malformed single digit and absence of the tibia
(Fig. 3B; see also Table S1 in
the supplementary material). The most proximal element of the hindlimb, the
stylopod (femur), was always normal. As similar distal truncations were
previously observed in chick limb Apical Ectodermal Ridge (AER) extirpation
studies (Summerbell, 1974), we
examined the consequences of Brg1 ablation on the AER. To this end,
we analysed the expression of Fgf8, which is both a marker of the AER
and a key mediator of its organizing functions along the proximodistal axis
(Moon and Capecchi, 2000
;
Sun et al., 2002
).
Fgf8 was similarly expressed in the AER of control and
Brg1ep-/-(c) forelimbs from E10 to E12, and in control and
Brg1ep-/-(c) hindlimbs at E10
(Fig. 3C, and data not shown).
However, at E10.5, Fgf8 expression in the
Brg1ep-/-(c) hindlimbs appeared diffuse, and was patchy as
well as drastically reduced at E11.5 (Fig.
3C). At E12, Fgf8 transcripts were no longer detected in
Brg1ep-/-(c) hindlimbs, in contrast to control hindlimbs
(data not shown). Histological analysis of E11.5 hindlimbs revealed that the
AER of Brg1ep-/-(c) fetuses was highly abnormal. Indeed,
cells in the AER region were smaller and much less densely packed than in
control foetuses. Their morphology was more similar to neighbouring ectodermal
cells (Fig. 3D). Thus, AER
formation and maintenance appear normal in forelimbs of
Brg1ep-/-(c) fetuses, whereas, in hindlimbs, the AER
formation is initiated, but is not maintained from E10.5 onwards, thus
resulting in selective hindlimb defects.
|
To investigate this barrier function, we first determined the diffusion of
the fluorescent dye Lucifer Yellow through the skin. As expected the dye was
retained in the upper layers of the stratum corneum of E18.5 control fetuses
(Matsuki et al., 1998),
whereas it diffused in mutant fetuses through the stratum corneum, and was
found in the dermis and hypodermis (Fig.
3E, and data not shown). That the barrier function was impaired in
the mutants was further supported in fetuses subjected to a whole-mount
permeability assay, based on endogenous ß-galactosidase activity of skin
at low pH (Hardman et al.,
1998
). As described, E18.5 control fetuses showed very little, if
any, X-Gal staining. By contrast, age-matched Brg1ep-/-(c)
fetuses stained strongly with X-Gal in most regions of the body surface
(Fig. 3F). Moreover, E18.5
Brg1ep-/-(c) mutant fetuses exhibited a sevenfold higher
trans-epidermal water loss (TEWL) in the dorsal and ventral region than
control littermates did, and, in contrast to control fetuses, lost 5-10% of
their body weight within 4-6 hours of cesarian delivery
(Fig. 3G and data not
shown).
|
Immunohistochemical analyses revealed that keratin (K)5 and K14 were similarly expressed in the basal layer of E18.5 control and mutant dorsal epidermis (data not shown). Moreover, the expression patterns of early (K1 and K10 in the spinous layer) and late (loricrin and fillagrin in the granular layer) markers of terminal differentiation were similar in control and mutant dorsal and ventral skin (data not shown). However, Nile Red staining performed on skin sections from E18.5 fetuses revealed that the surface lipid distribution was impaired in mutant fetuses. Indeed, while the neutral lipids formed a yellow-colored dense, continuous ribbon on top of the cornified layer in control fetuses, they were unevenly distributed along the cornified layer of the mutant epidermis (Fig. 5A, compare parts a and b).
|
Taken together, these results demonstrate that the lack of BRG1 in keratinocytes of the forming epidermis strongly impaired the formation of the skin permeability barrier.
Temporally controlled Brg1 ablation during epidermis formation
To investigate at which developmental stages BRG1 was required for hindlimb
development and skin barrier formation, we performed spatiotemporally
controlled Brg1 ablation, using
K14-Cre-ERT2(tg/0) transgenic mice that express the
tamoxifen (Tam)-activatable Cre-ERT2 recombinase under the control
of the human K14 promoter. We previously reported that, upon Tam
treatment of this transgenic line, efficient Cre-mediated excision of floxed
genes can be induced in keratinocytes of the basal cell layer of the adult
epidermis and in the outer root sheath (ORS) of the hair follicle
(Indra et al., 2000;
Li et al., 2000
). To
characterise ligand-dependent Cre-mediated recombination in the developing
epidermis, K14-Cre-ERT2(tg/0) transgenic males were bred
with ROSAfl/+ reporter females. embryos expressed
ß-galactosidase at E10.5 in the surface ectoderm of the posterior region,
in fore- and hindlimbs, and in pharyngeal arches
(Fig. 6A). To analyse
Cre-mediated recombination at later stages, 0.1 mg tamoxifen was daily
administered to females from either E9.5 to E11.5, E9.5 to E13.5, or E12.5 to
E16.5, and fetuses were recovered at E12.5, E14.5 and E18.5, respectively.
Skin sections revealed ß-galactosidase activity in most, if not all,
epidermal keratinocytes of the
K14-Cre-ERT2(tg/0)/ROSAfl/+ fetuses
analysed. By contrast, no X-Gal staining was observed in
K14-Cre-ERT2(tg/0)/ROSAfl/+ fetuses
from oil (vehicle)-treated females (Fig.
6A,B). These results show that Tam treatment of females during
gestation efficiently induced Cre activity in the surface ectoderm and
epidermal keratinocytes of fetuses, at various stages of epidermis
formation.
|
Like Brg1ep-/-(c) fetuses, E18.5 Brg1ep-/-(ia) mutant fetuses (Tam administration from E9.5 to 13.5) exhibited severe hindlimb and tail malformations (whereas forelimbs were unaffected), and died within 4-6 hours of cesarian delivery (Fig. 7A, part b, and data not shown). Moreover, skin defects similar to those of Brg1ep-/-(c) fetuses (Fig. 4A-C), i.e. flattening of skin, absence of corneodesmosomes, defects in the alignment of lamellar bodies were observed (data not shown). In marked contrast, when Tam was administered to females from 12.5-16.5 days of gestation, hindlimbs and tails were unaffected in the corresponding E18.5 Brg1ep-/-(ib) fetuses (see Fig. 7A, part d), which, however, exhibited impaired skin barrier function, lost 5-10% of their body weight within 4-6 hours of cesarian delivery, and died (Fig. 7D,E, and data not shown). These Brg1ep-/-(ib) mutant fetuses exhibited similar skin defects as Brg1ep-/-(c) and Brg1ep-/-(ia) mutants (Fig. 7B,C, and data not shown). Importantly, E18.5 Brg1L2/L2 fetuses from Tam- or vehicle-treated females had no limb and skin defects (see Fig. 7A, parts a and c, and data not shown).
|
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E18.5 Brm-/-/Brg1ep-/-(ib) fetuses
had normal fore- and hindlimbs, and their skin was formed
(Fig. 8A,B, and data not
shown). Moreover, they exhibited a similar basal cell proliferation rate to
control and Brg1ep-/-(c) fetuses, as determined by
immunohistochemical detection of Ki67 [a nuclear protein expressed in
proliferating cells (Schlüter et al.,
1993)] (Fig. 8C,D).
However, their spinous and granular cells displayed swollen cytoplasm and
nuclei, and only one to two cornified cell layer(s) were present
(Fig. 8B,E, and data not
shown). Moreover, their skin permeability barrier function was more
affected than that of Brg1ep-/-(ib) fetuses
(Fig. 8F,G). Thus,
keratinocytes lacking both BRM and BRG1 can proliferate and differentiate to
some extent, and even though BRM does not play any essential role in
suprabasal cells, it is partially redundant with BRG1 in these cells.
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Discussion |
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Conversely, keratinocytes lacking Brg1 undergo normal
stratification, but their late terminal differentiation is severely impaired.
Apparently normal lamellar granules (LG) containing lipid discs were present
in Brg1-null granular cells, but extrusion of their lipid content at
the interfaces between granular and cornified cells was altered, resulting in
disorganised lipid multi-lamellar structures in the stratum corneum, and
uneven neutral lipid distribution. This defective processing might be caused
by an altered lipid composition and/or protein content in the lamellar
granules. Moreover, the size and number of corneodesmosomes, which physically
hold the cells together in the stratum corneum, was markedly reduced, and thus
might be the basis of a reduced cohesion of the stratum corneum. The decrease
in corneodesmosome density in the lower cornified layers might result from an
impaired transfer of proteins from the lamellar granules into desmosomes,
including corneodesmosin (Lundstrom et
al., 1994), whose transcripts were reduced in Brg1 mutant
mice. As transglutaminase 1 (TGase1) and transglutaminase 3
(TGase3) transcripts were also reduced in fetal Brg1 mutant
skin, crosslinking of proteins might also be impaired in the statum corneum.
Together, these defects account for the defective skin permeability barrier
function and lead to an early postnatal death.
|
Interestingly, keratinocyte proliferation and stratification occur normally in Brm-/-/Brg1ep-/-(ib) fetal skin, in which Brg1 is ablated in Brm-/- keratinocytes after E12.5, which indicates that SWI2/SNF2 protein complexes are not essential for basal cell proliferation and/or early differentiation. However, even though the epidermis of Brm null fetuses appears normal, more severe defects were observed in suprabasal cells of Brm-/-/Brg1ep-/-(ib) fetuses than in those of Brg1ep-/-(ib) fetuses. Thus, BRM can partially substitute for BRG1 functions in these cells.
Taken together, our data indicate that BRG1-selective control of gene expression in the epidermal keratinocyte lineage is restricted to its terminal stages of differentiation, and that BRM is partially redundant with BRG1.
Brg1 ablation in the limb ectoderm induces hindlimb defects
We have shown here that Brg1 ablation in the limb ectoderm of
K14-Cre/Brg1L2/L2
[Brg1ep-/-(c)] embryos induces severe hindlimb defects.
The AER is known to be essential at early stages of limb development to
establish appropriately sized progenitor cell populations for the proximal
(stylopod), middle (zeugopod) and distal (autopod) segments. Surgical removal
of the AER from an early developing limb bud results in the loss of almost all
limb structures, whereas its removal at later stages results in progressively
more distal losses, while proximal structures are unaffected
(Summerbell, 1974). Similarly,
early genetic ablation of both Fgf4 and Fgf8 results in a
complete failure of limb development, whereas later inactivation of
Fgf4 and Fgf8 results in the production of all segments
along the proximodistal axis, but with distal segments that are reduced in
size and number (Sun et al.,
2002
).
In Brg1ep-/-(c) conditional mutants, in which
Brg1 was ablated in the limb surface ectoderm around E11, the
skeletal elements derived from the proximal hindlimb segment developed
normally, but those derived from the zeugopod and autopod were severely
hypoplastic or absent. Interestingly, the expression of Fgf8, a key
factor for AER function, which was initially similar in the hindlimb buds of
control and Brg1ep-/-(c) embryos, was progressively
affected by E10.5, and disappeared in Brg1ep-/-(c) embryos
at E12. In this respect, we note that although Fgf8 expression was
already severely altered at E10.5, BRG1 protein levels were strongly decreased
by E11 only. Thus, it appears that a moderate reduction of BRG1 levels could
be sufficient to impair FGF8 production, and therefore AER maintenance.
Altogether, our results show that BRG1 activity is essential in the hindlimb
ectoderm for the maintenance of the AER, most probably through the regulation
of Fgf8 signaling (Sun et al.,
2002).
Surprisingly, Fgf8 was normally expressed in
Brg1ep-/-(c) forelimbs, which developed properly, even
though the expression of BRG1 was sharply decreased in about 70% of the cells
of the forelimb surface ectoderm at E11.0, and was undetectable at E11.5. Thus
Brg1 might only be required for forelimb AER maintenance and
Fgf8 expression during a limited time period, before E10.5-E11.5.
Interestingly, temporally controlled Brg1 ablation in the surface
ectoderm by Tam-treatment of females from E9.5 to E13.5 similarly resulted in
hindlimb, but not forelimb defects. By contrast, ablation of Brg1 in
the forming epidermis at later time (Tam treatment from E12.5 to E16.5) did
not induce any limb defect, indicating that Brg1 plays a crucial role
during limb morphogenesis in a narrow temporal window. The absence of forelimb
abnormalities probably reflects differences in the timing of Brg1
ablation in the forelimb and in the hindlimb, rather than a differential
involvement of Brg1 in fore- and hindlimb development. Forelimb bud
formation is induced at E9, i.e. 24 hours earlier than hindlimb bud formation
(Kaufman and Bard, 1999), and
our data indicate that during this time period Cre-mediated DNA excision is
more efficient in the surface ectoderm of the posterior part of the
embryo.
Conclusion
Taken together, our results indicate that Brg1 controls
selectively the expression of genes involved in the epithelial-mesenchymal
interactions required for limb patterning
(Byrne et al., 2003) and in
terminal keratinocyte differentiation during epidermal histogenesis. As it is
the case for undifferentiated F9 embryonal carcinoma cells and in
peri-implantation embryos (Bultman et al.,
2000
; Sumi-Ichinose et al.,
1997
), BRM, which is dispensable for epidermis and limb formation,
cannot functionally replace BRG1 in these processes. However BRM can partially
substitute for BRG1 in keratinocytes undergoing terminal differentiation.
Importantly, as keratinocytes lacking both BRM and BRG1 in developing fetuses
proliferate and undergo early differentiation, it appears that some cellular
programs do not require SWI2/SNF2-containing chromatin remodelling complexes.
Rather, they might be controlled by ATP-dependent nucleosome remodelling
complexes containing other members of the SNF2 subfamilies, such as ISWI or
Mi-2. Last but not least, the present study demonstrates that, using
Cre-ERT2-mediated recombination, cell-specific targeted somatic
mutations can be created at various times during the development of the mouse
embryo, making possible to dissect gene function throughout morphogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/20/4533/DC1
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