1 INSERM U636, Centre de Biochimie, Faculté des Sciences, Nice,
France
2 Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany
3 Johannes-Müller-Institut für Physiologie,
Charité-Universitätsmedizin Berlin, Germany
Author for correspondence (e-mail:
holger.scholz{at}charite.de)
Accepted 5 January 2005
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SUMMARY |
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Key words: Wt1, Olfactory epithelium, Mash1, Alternative splicing, Neuron development
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Introduction |
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At least 24 different Wt1 proteins are generated by the combination of
alternative mRNA splicing (Haber et al.,
1991; Gessler et al.,
1992
), the use of variable translation start sites
(Bruening and Pelletier, 1996
;
Scharnhorst et al., 1999
), and
RNA editing (Sharma et al.,
1994
). Among the various gene products, alternatively spliced exon
5 encodes 17 amino acids, and the use of two alternative splice donor sites at
the end of exon 9 leads to the insertion/omission of a tripeptide
(lysine-threonine-serine, KTS) between zinc fingers 3 and 4 of the Wt1
molecule (Haber et al., 1991
).
The proteins, which are encoded by the alternatively spliced Wt1
forms, are designated as Wt1(-KTS) and Wt1(+KTS), respectively. Wt1(-KTS) has
been reported to function as both, an activator and a repressor of gene
transcription (Englert et al.,
1995a
; Lee et al.,
1999
) (reviewed by Menke et
al., 1998
; Scharnhorst et al.,
2001
). By contrast, the results of several studies suggested that
the +KTS isoforms, which comprise more than 50% of the Wt1 proteins
(Haber et al., 1991
;
Hammes et al., 2001
), could
play a role in mRNA processing (Larsson et
al., 1995
; Englert et al.,
1995b
; Ladomery et al.,
1999
). Thus, the Wt1(+KTS) products colocalized
with and bound to the nuclear splicing factor U2AF65
(Davies et al., 1998
).
Moreover, computer modelling (Kennedy et
al., 1996
) and in vitro studies
(Caricasole et al., 1996
)
testified that the +KTS proteins bind to RNA, whereas the -KTS isoforms
preferentially interact with DNA sequences. However, bona fide downstream
targets of the Wt1(+KTS) products have not been identified yet. In an effort
to analyse the roles of different Wt1 proteins during development, mouse lines
with selective ablation of either of the two splice insertions were generated.
Although removal of exon 5 caused no obvious phenotypic abnormalities
(Natoli et al., 2002
),
selective ablation either of the Wt1(-KTS) or the
Wt1(+KTS) product revealed distinct functions of these
proteins during gonad and kidney formation
(Hammes et al., 2001
). The
specific roles of the -KTS and +KTS proteins, which are conserved among
vertebrates (Kent et al.,
1995
; Miles et al.,
1998
), have not been analysed in neuronal tissues yet.
The present study served a twofold purpose. First, we aimed to further establish a role for Wt1 in neuronal development through identifying novel sites of Wt1 expression in the immature CNS. Second, by comparing the phenotype of mouse embryos with selective inactivation either of the -KTS or the +KTS variant, we made a first step towards understanding specific functions of alternatively spliced Wt1 gene products in the developing brain.
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Materials and methods |
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Histology and immunohistochemistry
Morphological studies were performed as described in detail elsewhere
(Wagner et al., 2002a;
Wagner et al., 2003
). Staged
embryos (morning of vaginal plug was considered E0.5) were fixed overnight at
4°C in paraformaldehyde (3% in PBS) and either embedded in paraffin wax
for Haematoxylin-Eosin (HE) staining or snap-frozen in pre-chilled isopentane
and then embedded in Tissue-Tek® OCT compound (Sakura Finetek,
Netherlands) for immunohistochemical analyses. Tissue sections (10 µm) were
cut and transferred onto gelatin-coated glass slides. The tissue sections were
permeabilized with 0.1% Triton X-100 in PBS and blocked by incubation for 1
hour in 10% normal serum (in PBS, 0.1% Triton X-100, 3% BSA), which was
obtained from the same species as the secondary antibody. Following treatment
(16 hours, 4°C) with primary antibody and 3x15 minutes washes in
PBS, the slides were incubated for 1.5 hours with biotinylated secondary
antibodies (1:150 dilutions in PBS, 1% BSA, Vector Laboratories) and
streptavidin-Cy3 complex (Sigma, Deisenhofen, Germany). The sections were
viewed under an epifluorescence microscope (Axiovert S100, Zeiss, Jena,
Germany), which was connected to a digital camera (Spot RT Slider, Diagnostic
Instruments), using the Metamorph V4.1.2 software (Universal Imaging). For
double immunostaining, the first antigen was detected using the Vector M.O.M.
immunodetection kit (Vector Laboratories) and streptavidin-Cy3 complex
followed by incubation with the second primary antibody and a Cy2-labelled
secondary antibody. Appropriate negative controls were made using normal sera
instead of primary antibodies. The following primary antibodies were used for
immunohistochemical analyses: Wt1 polyclonal antibody from rabbit diluted
1:150 (C-19, sc-192, Santa Cruz Biotechnology, Heidelberg, Germany), Wt1
monoclonal antibody from mouse diluted 1:100 (clone 6F-H2, MAB4234, Chemicon),
Pou4f1 mouse monoclonal antibody diluted 1:150 (14A6, sc-8429, Santa Cruz
Biotechnology), Pou4f2 polyclonal antibody from rabbit diluted 1:150 (C-13,
sc-6026, Santa Cruz Biotechnology), Ki-67 polyclonal antibody from goat
diluted 1:150 (M-19, sc-7846, Santa Cruz Biotechnology), neurogenin 1
polyclonal antibody from rabbit diluted 1:150 (AB5680, Chemicon, Temecula,
CA), Mash1 rabbit polyclonal antibody diluted 1:150 (AB5696, Chemicon), Mash1
monoclonal antibody from mouse diluted 1:100 (clone 24B72D11.1, 556604, BD
Biosciences), NCAM polyclonal antibody from rabbit diluted 1:1000 (AB5032,
Chemicon) and GFAP polyclonal antibody from rabbit diluted 1:1000 (AB5804,
Chemicon).
Detection of apoptotic cells
Apoptotic cells were localized in the olfactory epithelia of
paraformaldehyde-fixed mouse embryos by TUNEL-labelling with the In Situ Cell
Death Detection Kit (Roche Molecular Biochemicals, Mannheim, Germany) as
described in detail previously (Wagner et
al., 2002a). Five 10 µm transverse sections of the olfactory
epithelium were obtained from each animal to mark the apoptotic cells. Five
animals were studied in each group at E18.5.
Cell culture
The human embryonic kidney cell line, HEK293 (ATCC CRL-1573), was purchased
from the American Type Culture Collection (ATCC). The cells were grown in
Dulbecco's modified Eagle's medium (Invitrogen GmbH, Karlsruhe, Germany)
supplemented with 10% FCS (Biochrom KG, Berlin, Germany), 100 IU/ml penicillin
(Invitrogen) and 100 µg/ml streptomycin (Invitrogen). The cells were split
twice per week at 80% confluence for routine maintenance. The
transfection procedure and the selection of clones with stable expression of
the Wt1(-KTS) and Wt1(+KTS) proteins is described elsewhere
(Wagner et al., 2001
).
Reverse transcription (RT) PCR
Total RNA was prepared from HEK293 cells using the Trizol reagent
(Invitrogen). The RNA pellet was dissolved in diethyl pyrocarbonate-treated
H2O at a concentration of 1 µg/µl. First-strand cDNA
synthesis was performed with 2 µg of total RNA using oligo(dT) primers and
superscript II reverse transcriptase (Invitrogen). One-tenth of the reaction
product was used for PCR amplification in a thermal cycler (GeneAmp PCR System
2400, Perkin Elmer) according to the following protocol: DNA denaturation at
94°C, primer annealing at 58°C, extension of double-stranded DNA at
72°C (32 cycles, each step lasting 30 seconds). The following primers were
used for PCR amplification: human GAPDH,
5'-AACAGCGACACCCACTCCTC-3' (forward primer) and
5'-GGAGGGGAGATTCAGTGTGGT-3' (reverse primer); human achaete-scute
complex-like 1 (ASCL1), 5'-GAACTGATGCGCTGCAAACGC-3'
(forward primer) and 5'-CGGCCATGGAGTTCAAGTCGT-3' (reverse primer);
mouse Wt1, 5'-ATCAGATGAACCTAGGAG-3' (forward primer) and
5'-CTGGGTATGCACACATGA-3' (reverse primer). The amplified DNA
sequences were 257 bp (GAPDH), 333 bp (ASCL1) and 269 bp
(Wt1) long.
SDS-PAGE
Total cell lysates from subconfluent cultures of HEK293 cells were prepared
in a buffer consisting of 8 M urea, 10% (v/v) glycerol, 1% SDS, 10 mM Tris, pH
6.8 supplemented with 1 x protease inhibitor cocktail (Roche Molecular
Biochemicals), 10 mM DTT and 1 mM vanadate. Protein (60 µg) was heated to
95°C for 3 minutes in Laemmli buffer (500 mM Tris-HCl, 100 mM DTT, 2% SDS,
0.1% bromophenol blue, 10% glycerol, pH 6.8) and run on a 10% polyacrylaminde
gel. The separated proteins were transferred onto polyvinylidene difluoride
membranes (Amersham Pharmacia Biotech, Freiburg, Germany) with the use of a
semidry blotting apparatus (BioRad, München, Germany). Non-specific
binding was reduced by incubating the membranes for 60 minutes at room
temperature in PBS, 5% Blotto (Santa Cruz Biotechnology), 0.05% Tween-20
(Serva, Heidelberg, Germany). Incubation with a polyclonal anti-Wt1 antibody
from rabbit (C-19, sc-846, Santa Cruz Biotechnology, 1:100 dilution in PBS, 5%
Blotto, 0.05% Tween-20) and polyclonal anti-Mash1 antibody from goat (C-16,
sc-13222, Santa Cruz Biotechnology, 1:100 dilution in PBS, 5% Blotto, 0.05%
Tween-20) was performed overnight at 4°C. After 3x15 minutes washes
in PBS, 0.05% Tween-20, incubation was performed at room temperature for 1
hour either with peroxidase-coupled goat anti-rabbit secondary antibody to
detect Wt1 or with rabbit anti-goat secondary antibody to detect Mash1
(1:1.000 dilution in PBS, 5% Blotto, 0.05% Tween-20). Following 3x15
minutes washes in PBS, 0.05% Tween-20, the reaction products were detected
with the enhanced chemoluminescence system (Amersham Pharmacia Biotech,
Freiburg, Germany). For further analysis, the blots were stripped with 0.2 M
glycine, pH 2.5, at 56°C for 30 minutes and reprobed with a goat
polyclonal antibody against ß-actin (1:500 dilution in PBS, 5% Blotto,
0.05% Tween-20, C-11, sc-1615, Santa Cruz Biotechnology).
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Results |
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Next, we addressed the question whether abnormal development of the olfactory epithelium in the Wt1(+KTS)-/- mutant mice was due to reduced cell survival and/or impaired proliferation of neuronal progenitor cells. A total of six tissue sections from five different E18.5 embryos in each group of the wild-type as well as the Wt1(+KTS)-/- and the Wt1(-KTS)-/- mice were analysed. The numbers of TUNEL-positive cells that were identified in the basal parts of the olfactory epithelium on each tissue slide (Fig. 4) were 32±7 in wild-type animals, 65±7 in the Wt1(-KTS)-/- mutants and 167±4 in embryos with lack of the Wt1(+KTS) product (ANOVA Test with Dunn post-hoc test, wild-type versus the Wt1(+KTS)-/- mutants, P<0.05). Furthermore, the number of Ki-67-positive cells was reduced in the olfactory epithelia of Wt1(+KTS)-deficient embryos compared with the wild-type and Wt1(-KTS)-/- mice at E18.5 (Fig. 4). Similarly, immunostaining of the proliferating cell nuclear antigen (PCNA) was weaker in mouse embryos with specific lack of the Wt1(+KTS) protein (not shown).
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A double-immunofluorescent staining procedure was applied to explore whether Wt1 and Mash1 are colocalized in cells of the developing olfactory epithelium. Remarkably, a proportion of cells expressed both proteins suggesting that Wt1 can possibly regulate Mash1 expression also in the developing olfactory epithelium in vivo (Fig. 6).
Different Wt1 splice variants are predominant during development of the retina and the olfactory epithelium
We have recently found that Wt1 is necessary for normal
development of the retina (Wagner et al.,
2002a). Complete inactivation of Wt1 caused severe
retinal defects consisting in an impaired proliferation of neuronal progenitor
cells and an apoptotic loss of ganglion cell precursors
(Wagner et al., 2002a
). To
distinguish which of the alternatively spliced Wt1 variants would be crucial
for the development of the neural retina, we analysed the ocular phenotype of
mice with specific lack either of the Wt1(-KTS) or the Wt1(+KTS) protein.
Compared with the wild-type embryos at E12.5, the retinas of the
Wt1(-KTS)-/- mutants were clearly thinner and contained
fewer cells (Fig. 7A).
Abnormalities of the developing ganglion cell layer became visible at E18.5 in
the Wt1(-KTS)-deficient embryos
(Fig. 7B). These retinal
defects were less severe in embryos with lack of Wt1(+KTS)
(Fig. 7), indicating that
normal formation of the retina depends mainly on the function of the Wt1(-KTS)
protein.
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Discussion |
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Induction of the olfactory sensory tissue, which originates from
ectodermally derived neurogenic placodes, is dependent on
mesenchymal/epithelial interaction in the developing forebrain
(LaMantia et al., 2000). Among
the molecules that have been implicated in the epithelial conversion of
mesenchymal cells in other organs is the product of the Wilms' tumour gene,
Wt1 (Kreidberg et al.,
1993
; Moore et al.,
1999
). Wt1 was originally identified by its mutational
inactivation in a subgroup of paediatric renal tumours (Wilms' tumours,
nephroblastomas) (reviewed by Hastie,
1994
). Subsequent studies revealed a crucial role for Wt1
in the formation of the genitourinary system
(Kreidberg et al., 1993
) and
other epithelial tissues of mesenchymal origin
(Herzer et al., 1999
;
Moore et al., 1999
). We have
recently discovered that Wt1 is also crucial for neurogenesis, in that the
retinas of Wt1-deficient mice failed to develop normally
(Wagner et al., 2002a
). Our
current findings extend the role of Wt1 in neuronal differentiation
by demonstrating that the formation of the olfactory epithelium is severely
disturbed in Wt1(+KTS)-deficient mice. Although the initial
formation of the olfactory sensory tissue in the Wt1(+KTS)
mutants seemed intact, defective morphology at E12.5 indicates a requirement
for Wt1 during the early stages of olfactory development. A reduced
proliferation of progenitor cells of the Wt1(+KTS)-deficient
embryos is suggested from the fewer Ki-67 positive cells in the olfactory
epithelium compared with wild-type and Wt1(-KTS)-/- mutant
mice. In addition, more TUNEL-positive cells were detected in the olfactory
epithelia of mice, which lacked the Wt1(+KTS) form, than in
normal and Wt1(-KTS)-deficient embryos. Thus, enhanced
apoptotic cell death may account, at least in part, for the defective
olfactory epithelia of embryos with inactivation of
Wt1(+KTS). Very similar observations have previously been
made in the developing neuronal retina of mice with disrupted Wt1
gene, which also displayed more TUNEL-positive cells than wild-type embryos
(Wagner et al., 2002a
), and in
the kidneys and gonads of Wt1-deficient mice
(Kreidberg et al., 1993
;
Hammes et al., 2001
). Thus,
apoptotic cell death in Wt1-/- embryos appears to occur
mainly in tissues that would express Wt1 in normal mice. It remains to be
established whether Wt1 rescues cells from apoptosis through a direct
anti-apoptotic action. Alternatively, and perhaps even more likely, Wt1 may
function as a regulator of cell differentiation, and lack of Wt1 will lead to
apoptosis due to a failure of normal cellular specification.
Altered formation of the olfactory epithelium in embryos with ablation of
the Wt1(+KTS) protein, is also reflected in their hypoplastic olfactory bulbs.
Notably, Wt1 could not be detected, at least by the means of
immunohistochemistry, in the olfactory bulbs of wild-type embryos.
Consequently, impaired olfactory bulb formation in the
Wt1(+KTS)-deficient embryos was secondary to their abnormal olfactory
epithelium rather than resulting from a cell-autonomous defect of olfactory
bulb cells. Interestingly, defects of the developing olfactory system became
apparent only in embryos, which lacked the Wt1(+KTS) splice
variant, but not in the Wt1(-KTS)-/- mutants. However,
embryos with ablation of Wt1(-KTS) had more severe retinal
abnormalities than the Wt1(+KTS)-deficient mice. Similar to
embryos with complete Wt1 knockout
(Wagner et al., 2002a), the
Wt1(-KTS)-/- retinas contained fewer progenitor cells in
addition to their failure to form a normal ganglion cell layer. Thus, the
development of the retina seems to depend mainly on the function of the
Wt1(-KTS) protein, which has been implicated in the control of gene
transcription. Accordingly, the Pou-domain factor Pou4f2 (formerly Brn-3b),
which is required for retinal ganglion cell development
(Gan et al., 1996
;
Erkman et al., 1996
) and whose
transcription is activated by Wt1(-KTS)
(Wagner et al., 2003
), was
virtually missing in the ganglion cells of the
Wt1(-KTS)-/- retinas.
Evidence has been provided that Wt1 proteins, which contain the +KTS splice
insertion, might act at a post-transcriptional level rather than functioning
as transcriptional regulators (Larsson et
al., 1995; Englert et al.,
1995b
; Davies et al.,
1998
; Ladomery et al.,
1999
; Laity et al.,
2000
). However, physiologically relevant in vivo targets of the
Wt1(+KTS) isoforms have not been identified yet. By comparing the gene
expression profiles in the olfactory epithelia of normal embryos and of mice
with lack of the Wt1(+KTS) variant, one may eventually
succeed in isolating potential downstream target molecules. A first candidate
gene for regulation by the Wt1(+KTS) protein could be the mammalian homologue
of achaete-scute complex, Mash1 (Ascl1). Expression
of Mash1, which encodes a basic helix-loop-helix (bHLH) transcription
factor, was reduced in the Wt1(+KTS)-deficient olfactory
epithelium. Mash1 is a crucial molecule for the proliferation and neuronal
specification of progenitor cells in the ventral telencephalon
(Casarosa et al., 1999
). Mice
with homozygous null alleles for Mash1 exhibited a severe reduction
of olfactory neurons due to impaired progenitor cell proliferation and
apoptotic cell death (Guillemot et al.,
1993
; Cau et al.,
1997
). Neurogenin 1, another proneural bHLH transcription factor,
is expressed at a later stage of olfactory progenitor development than Mash1
(Cau et al., 1997
). Remarkably,
most cells in the olfactory epithelium of Mash1-null mutant embryos
failed to produce neurogenin 1, indicating that Mash1 is required for normal
expression of neurogenin 1 (Cau et al.,
1997
). Consistently, we found that both proteins, Mash1 and
neurogenin 1, were only weakly expressed in the olfactory epithelia of
Wt1(+KTS)-deficient embryos. It remains to be clarified
whether the Wt1(+KTS) splice product provides a signal for
the proliferation and/or survival of Mash1-positive olfactory progenitor
cells, or whether it may even stimulate the expression of Mash1 more directly.
The latter possibility is supported by our observation that Mash1 was enhanced
by forced expression of Wt1(+KTS), but not of the -KTS variant, in cultured
cells derived from human embryonic kidney. Recent findings suggest that the
HEK293 cells, which we used, resemble neurons rather than renal epithelial
cells (Shaw et al., 2002
).
Their neuronal origin, which is indicated by the expression of several
neuron-specific marker proteins in HEK293 cells
(Shaw et al., 2002
), could be
a reason for the strong increase of Mash1 in response to forced Wt1
expression. The molecular mechanism by which Wt1(+KTS) activates the
expression of Mash1 remains to be further clarified in future
studies. The lack of stimulation of the Mash1 promoter by Wt1 argues in favour
of either a post-transcriptional interaction between Wt1(+KTS) and
Mash1, or simply signifies that additional cis-regulatory elements,
which were not contained in our promoter construct, are required. Remarkably,
a significant fraction of Wt1-immunopositve cells in the developing olfactory
epithelium of wild-type embryos also contained Mash1. This observation points
to the possibility that Wt1 can activate the expression of Mash1 not only in
cultured cells, but also in neuronal progenitor cells in vivo. Taken together,
our findings demonstrate that a splice variant of the Wilms' tumour gene
Wt1 plays a crucial role during development of the olfactory system.
The phenotype of mouse embryos with lack of the Wt1(+KTS)
product reveals a requirement of this protein for the proliferation and
survival of olfactory progenitor cells. On the contrary, formation of the
neuronal retina mainly depends on the Wt1(-KTS) protein, which acts as a
transcription factor. In conclusion, neuron formation in the embryonic retina
and the olfactory epithelium requires different functions exerted by
alternatively spliced Wt1 products.
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ACKNOWLEDGMENTS |
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Footnotes |
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