Departamento de Neurobiología del Desarrollo, Instituto Cajal, CSIC, Dr Arce 37, Madrid 28002, Spain
* Author for correspondence (e-mail: bovolenta{at}cajal.csic.es)
Accepted 7 March 2003
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Summary |
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Key words: Chick, Eye development, Retina ganglion cells, Wnt signalling, GSK3ß
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Introduction |
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Wnt activities turn on distinct signalling cascades. In all cases, Wnt
signalling is initiated by WNT binding to Frizzled (Fz), a family of G-coupled
transmembrane receptors (Liu et al.,
2001; Winklbauer et al.,
2001
). In the canonical pathway, receptor-ligand interaction leads
to a protein complex-mediated inhibition of the glycogen synthase kinase3b
(GSK3ß) with a consequent cytoplasmic accumulation of ß-catenin,
which activates Wnt target genes after nuclear translocation
(Wodarz and Nusse, 1998
).
Alternatively, Fz activation appears either to stimulate intracellular
Ca2+ release, leading to activation of protein kinase C
(Kuhl et al., 2000
;
Winklbauer et al., 2001
), or
to activate the planar cell polarity pathway that involves Jun-kinase mediated
transcription (Heisenberg et al.,
2000
; Strutt,
2001
; Weber et al.,
2000
). The complex signalling mechanisms of WNTs are further
controlled in the extracellular space by a number of soluble molecules
including the family of Secreted Frizzled Related Proteins, SFRPs
(Jones and Jomary, 2002
)
As their name indicates, SFRPs are secreted proteins that share structural
homology with the extracellular cystein rich domain (CRD) of the Fz domain
(Finch et al., 1997;
Melkonyan et al., 1997
;
Rattner et al., 1997
;
Wang et al., 1997
), which in
Fz is necessary and sufficient for WNT binding
(Wodarz and Nusse, 1998
). The
structural homology of SFRPs with the Wnt receptors and their widespread
expression, complementary in several locations to those of Wnts, have
suggested that SFRPs could act as competitive antagonists of WNT-mediated
ß-catenin signalling, binding and preventing Wnt interaction with the Fz
receptors (Leyns et al., 1997
;
Wang et al., 1997
;
Xu et al., 1998
). However,
both biochemical and functional analyses of SFRP activities have demonstrated
that SFRP mode of action might be more elaborate than that originally
envisaged. SFRP1 can form heterodimeric complexes with other members of the
family or with Fz receptors, suggesting alternative mechanisms of interfering
with the Wnt pathway (Bafico et al.,
1999
). Furthermore, expression studies in Xenopus embryos
and analysis in mammalian cell lines indicated that different SFRPs have
opposing activities (Bradley et al.,
2000
; Melkonyan et al.,
1997
; Pera and De Robertis,
2000
) and few of them may function independently of any known
WNTs, suggesting that SFRPs might regulate developmental processes in an
autonomous way (Bradley et al.,
2000
).
Recently, we and others have isolated and studied the distribution of the
chick homologue of Sfrp1 (Esteve
et al., 2000; Terry et al.,
2000
). cSfrp1 has a widespread and dynamic expression in
the developing chick embryo and is abundantly localized during the processes
of neuronal differentiation in distinct CNS regions, including the eye.
Initiation of eye development appears to require the Wnt signalling cascade
(Heisenberg et al., 2001
;
Rasmussen et al., 2001
).
However the precise role of Wnt, Fz receptors and SFRPs, all expressed in the
eye at later stages of development
(Deardorff and Klein, 1999
;
Jasoni et al., 1999
;
Jin et al., 2002
;
Sumanas and Ekker, 2001
), has
yet to be defined.
With in vitro and in vivo studies, we show here that SFRP1 modulates cell differentiation of the chick retina promoting the generation of retinal ganglion cells (RGC) and cone photoreceptor cells, at the same time as decreasing the number of amacrine cells. Because retina cells have a low basal ß-catenin transcriptional activity that is not modified by SFRP1, we propose that SFRP1 contributes to retina neurogenesis with a mechanism that does not require its interference with a ß-catenin-dependent Wnt-Fz interaction.
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Materials and Methods |
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Isolation of cSfrp1 cDNAs and in situ hybridisation
cSfrp1 isolation and in situ hybridisation procedures have been
previously described (Esteve et al.,
2000). The accession number for cSfrp1 is AJ404652 in the
Nucleotide Sequence Databases.
Expression and purification of chick SFRP1 protein and its
derivatives
The coding sequence of Sfrp1 was cloned in the pCDNA3.1 eukaryotic
expression vector (Invitrogen, Carlsbad, CA), containing a myc/his-tag. The
resulting construct coded for the SFRP1 protein in frame with the tag in its
C-terminus. Subconfluent MDCK cells, grown in Dulbecco's modified Eagle's
medium (DMEM; Gibco, BRL, Paisley, UK) supplement with 10% FCS were
transfected with 5 µg of Sfrp1 expression plasmid or with the
empty vector, using the Gene PORTER kit (Gene Therapy Systems, San Diego, CA).
Transfected cells were selected with Geneticine (500 µg/ml) for three
weeks. Individual G418-resistant clones were isolated and expression of the
recombinant protein was determined by immunoblots with an anti-myc mAb (clone
9E10). The myc/his-SFRP1 was partially purified from conditioned medium (CM)
using a Ni-NTA agarose (Qiagen, Valencia, CA) column. CM from MDCK transfected
with the empty vector was processed in parallel as control. The agarose was
washed and equilibrated in 50 mM phosphate buffer containing 10 mM imidazole.
SFRP1 protein was eluted with 100 mM imidazole, as reported
(Uren et al., 2000). The
control and SFRP1 eluted fractions were analysed by silver staining and
western blotting.
Dissociated retinal cell culture
The central portion of the neural retinas from embryonic day (E)5 chick
embryos were dissected free of pigmented epithelium and dissociated as
described (Bovolenta et al.,
1996). Cells were plated in 33 mm dishes coated with poly-D-lysine
(20 µg/ml; Sigma) and cultured in DMEM supplemented with N2 nutrients
(Gibco BRL) alone or diluted 1:1 with CM from MDCK/control or MDCK/SFRP1. For
immunostaining, cultures were fixed in methanol containing 10% DMSO for 15
minutes at room temperature.
dsRNA-mediated interference
The templates used for RNA synthesis were either a linearised plasmid
containing hGFP, as a control, or the products of PCR reactions using primers
designed to amplify the N-terminal (nucleotide 75 to 410) or the C-terminal
(nucleotide 506 to 901) regions of chick Sfrp1. The products were
purified using GFX PCR DNA and Gel Purification Kit (Amersham Pharmacia,
Buckinghamshire, UK). Each primer contained a T7 promoter sequence on its
5' end such that sense and anti-sense RNAs, in an already annealed
conformation, can be synthesized simultaneously from a single PCR-derived
template using T7 RNA polymerase
(Kennerdell and Carthew,
1998). Following removal of template with DNAseI, RNAs were
purified using RNeasy Mini Kit (Qiagen). dsGFP RNA was generated from ssRNAs
synthesized using T7 and SP6 polymerase. The sense and anti-sense GFP RNAs
were mixed in annealing buffer (10 mM Tris, pH 7.4, 0.1 mM EDTA) heated at
68°C for 10 minutes and incubated at 37°C for 4 hours. The specificity
of the dsRNA, designed against the N-terminal or C-terminal portion of
Sfrp1, was tested using MDCK cells transfected with
myc-Sfrp1. The three dsRNAs were either electroporated or added to
the dissociated cell suspension (2.5 µg/6x105 cells).
After incubation cells were seeded, cultured for 24 hours and harvested. The
ability of Sfrp1 dsRNAs to interfere with myc-Sfrp1 mRNA and protein
levels were determined by PCR analysis and western blot of the CM of control
and dsRNA treated cells using anti-myc mAb. The Cter Sfrp1 dsRNA
resulted the most effective and was used in the experiments involving retina
cells. In each experiment, the level of endogenous retina Sfrp1 mRNA
was monitored in dsRNA treated, control treated and untreated cultures using
time-course PCR analysis. In all experiments, Sfrp1 Cterm dsRNA
reduced the mRNA level to about 60% of controls.
Reporter assays
E5 dissociated central retinal cells, prepared as described above, were
seeded in 24-well plates and transfected 3 hours later using the Gene PORTER
kit (Gene Therapy Systems). In each case the 2.5 µg/well of total DNA
contained 200 ng of a plasmid constituted by a Lef-1 responsive luciferase
reporter composed of four copies of the wild-type Lef-1 responsive element (or
the mutated element) upstream of the prolactine minimal promoter driving
luciferase expression (generated and kindly provided by Drs Gutierrez and M.O.
Landázuri, Hospital de la Princesa, Madrid) and 50 ng of pRL-TK
(Promega, Madison, WI) together with variable amounts of the effector plasmids
or the empty vector. Twenty-four hours after the transfection, the luciferase
activities were determined using a dual-luciferase assay system (Promega). The
LEF-1 reporter luciferase activity was normalized with Renilla
luciferase activity to account for transfection efficiency. The experiment was
repeated four times in duplicate.
Explant electroporation
Retina explants from E5 central retinas were suspended in PBS containing
100 ng/µl of DNA (pCSII-hGfp of pCDNA3.1-Sfrp1) and
electroporated with the following conditions: four pulses of 50 milliseconds
length, 508 milliseconds frequency, 20 V. After electroporation explants were
grown in suspension for 13 hours in DMEM/N2. Timing of initial protein
expression was determined by appearance of GFP fluorescence using a Leica,
MZ-FLIII macroscope.
Western blotting of dissociated retina cells or explant culture
Retina cultures were collected at 20 minutes, 3, 5 and 24 hours in PBS.
Explants were harvested approx 5 hours after detection of GFP expression.
Tissue was lysed in a buffer composed of 15 mM Tris, pH 8, 60 mM KCl, 15 mM
NaCl, 2 mM EDTA, 1% Nondiet-P40, 1 mM Na orthovanadate, 100 mM NaF and
proteases inhibitors. Samples were boiled in Laemmli's buffer and separated by
SDS-PAGE, transferred to nitrocellulose and probed with the following
antiserum: phospho-GSK3ß (Ser9; Cell Signalling Technology, Beverly, MA),
phospho-GSK3ß (Tyr216; Cell Signalling Technology) and -tubulin
(Sigma) or with a mAb anti-GSK3ß (Transduction Laboratories, Exeter, UK).
For detection of cytosolic ß-catenin levels, cells were harvested by
lysis in hypotonic solution (10 mM Tris buffer, pH 7.4; 200 µM
MgCl2, 1 mM PMSF, 10 µg/ml leupeptine and 10 µg/ml
aprotinine). Membrane and cytosolic fractions were separated by
ultracentrifugation at 100,000 g for 30 minutes.
Nitrocellulose membranes of the fractionated samples were obtained as above
and probed with anti-ß-catenin mAb (Transduction Laboratories).
Immunoreactive bands were detected with the ECL system
(Amersham-Pharmacia).
Retrovirus production and embryo infections
cSfrp1 or the dominant-negative form of the Xenopus
GSK3ß [dnGSK3ß; K85R abolishing kinase activity
(Dominguez et al., 1995)] were
cloned into the RCAS(B) vector and viruses were prepared as described
(Fekete and Cepko, 1993
).
Alkaline phosphatase-RCAS(B) viruses were used as control. Concentrated viral
suspensions (approx 108 cfu/ml) were injected into the optic
vesicles of stage 10-11 embryos. Embryos were harvested at E6 or E10, fixed in
4% paraformaldehyde and sectioned on a cryostat at 16 µm of thickness.
Immunohistochemistry
Staining of cultures and tissue sections was performed following standard
procedures. The antibodies used are mAb anti-tubulinß-III (Tuj-1, MEDPASS
S.a.r.l., Gran Duché de Luxembourg), rabbit polyclonal
anti-phospho-histone 3 (Upstate Biotechnology, Lake Placid, NY), a mitosis
marker (Mahadevan et al.,
1991), mAb anti-neurofilament 145 kDa protein (CHEMICON,
Ternecula, CA), anti-Brn3 (Santa Cruz Biotechnology, Santa Cruz, CA),
anti-cellular retinoic acid protein 1 (CRABP1; Affinity Bioreagents, Golden,
CO) and anti-islet1 guinea-pig antiserum (gift of T. Jessell, Columbia
University, NY). The mAbs anti islet-1, anti-Pax6, and anti-visinin developed
by T. Jessell, A. Kawakami and C. Cepko, respectively, were obtained by the
Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA).
For cell culture staining, secondary antibodies were biotin-conjugated anti-mouse or anti-rabbit IgG followed by Alexa-594- or Alexa-488-conjugated streptavidin (Molecular Probes, Eugene, OR). For tissue section staining, biotinylated-secondary antibodies were followed by peroxidase-coupled streptavidin (Jackson Laboratories, West Grove, PA) and AEC system chromogenic reaction (Lab Vision Corporation, Fremont, CA). Cultures were counterstained with BisBenzimide (HOESCHT no. 33342; Sigma).
Statistical analysis
Quantitative data were obtained by counting in each case the number of
Hoescht and immuno-positive cells present in a field of 0.038 mm2.
Each experiment was performed in duplicate at least four times; in each case
12 randomly chosen fields were analysed. Data are expressed as
mean±s.e.m. The significance of the differences among groups was
evaluated by unpaired Student's t-test (GraphPad Prism).
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Results |
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Production and purification of secreted SFRP1
To address the functional activity of SFRP1, we generated a source of
soluble SFRP1 protein. The MDCK cell line was stably transfected with an
expression vector containing the full coding sequence of myc-tagged
Sfrp1 or with the vector alone. To optimise the production of SFRP1,
single-cell-originated clones were isolated and tested for their ability to
secret the tagged protein. Although SFRP1-myc was detected in the cell line
extracts (not shown), the majority of the protein was released in the culture
medium of several of the isolated clones, as shown by immunoblots
(Fig. 2A). Clones 2, 3 and 10
secreted the highest yields of SFRP1 into the media and were used in the
subsequent studies (MDCK/SFRP1). The culture media from cells transfected with
the empty vector or media from clones unable to secrete the protein (i.e.
clone 7, Fig. 2A) were used as
controls (MDCK/control).
|
Purification of SFRP1 from the conditioned medium (CM) was attempted using
an affinity chromatography on a Ni2+ charged agarose column. This
single step procedure provided only a partial purification of the protein as
assessed by silver staining of the recovered fraction and comparison with
MDCK/control CM processed in parallel (Fig.
2B). An additional purification step on a heparin-agarose column,
as previously described (Uren et al.,
2000), did not improve the protein purification when compared with
the control (not shown). The CM or the affinity semi-purified protein were
used in subsequent studies aimed at defining the activity of SFRP1 during
chick neural retina development.
SFRP1 induces differentiation of retinal neuroepithelial cells
Initial functional studies were performed on dissociated retinal cultures,
where the direct response to SFRP1 could be assessed on each individual cell.
The order of retina cell type generation is conserved in vertebrates
(Livesey and Cepko, 2001), and
retinal ganglion cells (RGC) and cone photoreceptors constitute the
postmitotic neurons present in an E5 chick retina
(Prada et al., 1991
). The
majority of the retina neuroepithelium is still undifferentiated and precursor
cells can proliferate and differentiate in culture. Thus, E5 retinas were
dissociated, plated on poly-D-lysine (PDL)-coated dishes and maintained in
defined medium for 24 hours. Under these conditions and compared with control
cultures grown in the presence of CM from MDCK/control, addition of MDCK/SFRP1
CM or its semi-purified fraction did not induce significant variations in cell
density (Fig. 3G) or in the
rate of apoptotic cell death (not shown) and mitotic cell division, as
determined by staining with the mitotic marker PH3
(Fig. 3G). However,
SFRP1-treated cultures showed an increase in cell differentiation, as assessed
by staining with antibodies against distinct differentiation markers:
islet1/2, LIM-domain containing proteins, expressed in the retina
predominantly by RGC (Austin et al.,
1995
), visinin, expressed by cone photoreceptors
(Hatakenaka et al., 1985
), and
two other general neuronal markers, the 145 kDa neurofilament (NF) protein and
Tuj1, a neuron-specific tubulinßIII, which is expressed soon after the
last mitotic division. Thus, in the presence of SFRP1, there was an increase
in the number of Tuj1- (Fig.
3G), NF- (Fig.
3A,B) and visinin-positive cells
(Fig. 3E-G) with respect to
controls. This difference was less pronounced when the number of
differentiated cells was determined by staining with anti-islet1/2 mAb
(Fig. 3C,D,G), possibly
reflecting the later onset of this marker in the process of differentiation,
as compared with Tuj1. Alternatively, other phenotypes may be included in the
pool of Tuj1-positive cells.
|
At E5, Sfrp1 is endogenously expressed in most of the retina central
neuroepithelium. Therefore, interference with its expression should at least
in part modify the basal cell differentiation normally occurring in the
control cultures. We employed double-strand (ds) RNA-mediated interference
(RNAi) to deplete endogenous SFRP1
(Kennerdell and Carthew,
1998). To assess the specificity of the designed dsRNA, dsRNA
directed against the N-terminal or C-terminal portion of Sfrp1 and a
control dsRNA against GFP were added to MDCK cells transfected with
myc-Sfrp1. The three dsRNAs were either electroporated or added to
the dissociated cell suspension. Cells were then seeded and harvested after 24
hours. The ability of Sfrp1 dsRNAs to interfere with myc-SFRP1
protein levels was determined by western blotting of the CM of control and
dsRNA treated cells using anti-myc antibodies. The Cter Sfrp1 dsRNA,
and to a lower extent the Nter Sfrp1 dsRNA, but not the Gfp
dsRNA, consistently reduced the protein level (30% reduction) of
Sfrp1 in the CM of treated cells
(Fig. 4A), without affecting
cell viability. PCR analysis showed that the decrease in protein levels
induced by the Sfrp1 Cter dsRNA was paralleled by a clear reduction
in the mRNA levels (Fig. 4B).
This measure was subsequently used to analyse the interference with the
endogenous expression of Sfrp1 in E5 retinal cells.
|
Cter Sfrp1 dsRNA significantly diminished the endogenous mRNA level of Sfrp1 (Fig. 4C) and interfered with the differentiation of both islet1/2-positive RGC and visinin-positive cone photoreceptors when compared with that observed in the presence of a control dsRNA (Fig. 4D), which effect, in turn, was undistinguishable from that of untreated cultures (data not shown). In no cases was modification in the viability of the cultures ever observed.
Altogether these data are consistent with the idea that SFRP1 specifically favours retina cell differentiation.
Specific neuron classes are altered by overexpression of SFRP1 in
ovo
Neuronal differentiation is inhibited by the Delta-Notch signalling
pathway, which requires cell-cell contact
(Artavanis-Tsakonas et al.,
1995). Therefore, we asked whether forcing the expression of SFRP1
in intact chick retinas had consequences similar to those observed in
dissociated cells. The replication competent retroviral vector RCAS was used
to overexpress SFRP1 in the optic vesicles of St10-11 embryos, when the entire
neuroepithelium is still undergoing proliferation. Embryos were allowed to
develop until E6, when the majority of RGC, amacrine and cone photoreceptor
cells have exit the cell cycle (Prada et
al., 1991
). Using cell-specific differentiation markers, we
analysed whether the number of RGC (islet1-positive), cone photoreceptors
(visininpositive) and amacrine cells [CRABP1-positive
(Henrique et al., 1997
)] was
modified in viral infected retina tissue
(Fig. 5A,B). Immunostaining of
consecutive sections clearly indicated that RCAS-Sfrp1 infection caused an
increase in the number of cones (Fig.
5G,H). Similarly, RGC were not only more densely packed in the RGC
layer but were also more frequent in the ventricular zone, as compared with
equivalent retina regions of uninfected contralateral eyes or of eyes from
RCAS-AP infected embryos (Fig.
5E,F). In RCAS-SFRP1 treated retinas, a larger number of migrating
neurons was also observed with antibodies against Tuj1
(Fig. 5C,D). On the contrary,
CRABP1-positive amacrine cells, which are generated slightly later than RGC
and cones, were less abundant than in controls
(Fig. 5I,J). Comparison of the
amount of islet1-, visinin- and CRABP1-positive cells in patches of infected
retinas with equivalent control regions demonstrated an increase of about 30
to 40% in both RGC and cones, paralleled by a significant decrease in amacrine
cells (Table 1A). Similar
variations were observed also at later stages of retina development (E10),
using additional differentiation markers such as Brn3 or Pax6
(Hitchcock et al., 1996
;
Liu et al., 2000
).
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|
In conclusion, SFRP1 overexpression favoured the production of RGC and cones, while decreasing the number of amacrine cells. These changes occurred without apparent modifications in the rate of apoptotic cell death (data not shown) or cell division, as determined by staining with the mitotic marker PH3 (Fig. 5K,L, and Table 1), or in the mRNA distribution of either Notch or Delta1 (data not shown). Infection with RCAS-AP, used to determine possible unspecific effect of the virus, did not alter retina differentiation as compared with uninfected controls.
Analysis of RCAS-SFRP1 infected embryos at the beginning of retina differentiation (E3-4) already showed an increase of both Tuj1 or islet1 positive cells in the central retina. However, infections in the retina periphery, where the wave of cell differentiation had not spread yet, did not induce the appearance of ectopic neurons, indicating that SFRP1 overexpression can modify cell differentiation but, on its own, cannot initiate it.
SFRP1-mediated effects do not involve modification in the
ß-catenin-mediated transcriptional activity
WNT-mediated cell differentiation signals through the canonical
ß-catenin-dependent pathway (Miller
et al., 1999; Patapoutian and
Reichardt, 2000
). SFRP1-induced phenotype in the retina could be
the result of SFRP1 interference with an endogenous Wnt-Fz interaction that
signals through the ß-catenin pathway. If this is the case, then, SFRP1
should modify the cytosolic ß-catenin levels and the
ß-catenin-dependent transcriptional activity of the retina. However,
time-course immunoblot analysis of the retina cells cultured in the presence
or absence of SFRP1 did not reveal significant variations in the content of
cytosolic ß-catenin (Fig.
6A), even 24 hours later (data not shown). We next measured the
activity of the ß-catenin-responsive Tcf-binding site (or of its mutated
version) coupled to the luciferase reporter gene in transiently transfected
retinal cells. This assay showed that chick retina cells have a low basal
ß-catenin-dependent transcriptional activity, since similar values were
obtained with both the wild type and the mutated version of the reporter
plasmid, in line with a recent report in zebrafish retina
(Dorsky et al., 2002
).
Furthermore, transfections of different doses of Sfrp1 were unable to
modulate this basal TCF-transcriptional activity. Nevertheless, Sfrp1
was able to decrease in a dose-dependent manner the 240-fold activation of the
TCF-dependent transcriptional activity induced by Wnt8
(Fig. 6B). These results
suggest that, in developing chick retina, canonical Wnt signalling is not
normally activated and, therefore, in the absence of ß-catenin-dependent
Wnt signalling, SFRP1 may exert its function with an alternative
mechanism.
|
GSK3ß might be an effector of SFRP1 activity in the chick
retina
The current model of the canonical Wnt signalling suggests that in the
presence of Wnts GSK3ß is inactivated. Interestingly, however, inhibition
of GSK3ß activity has been also associated with an increase of neuron
differentiation in both Drosophila
(Bourouis et al., 1989) and
Xenopus embryos (Marcus et al.,
1998
; Moore et al.,
2002
). Therefore, we have asked whether SFRP1 activity could
involve inhibition of GSK3ß, hypothesising that Sfrp1 mechanism of action
might be different from that of an extracellular Wnt antagonist.
To this end, we used RCAS-mediated ectopic expression of the
dominant-negative form of the Xenopus GSK3ß [dnGSK3ß
(Dominguez et al., 1995)],
with an experimental design similar to that described for SFRP1
overexpression. In these experiments we observed a retina phenotype that
closely matched that of SFRP1 gain-of-function. Statistical analysis showed
that, without apparent modifications of the mitotic rate, RGC and cone
photoreceptors increased whereas amacrine cells decreased
(Table 1B), with percentages
that were remarkably similar to those obtained with SFRP1 overexpression
(compare Table 1A with 1B).
Furthermore, in retina cells, transfection of the dnGSK3ß was unable to
increase endogenous ß-catenin-dependent transcription
(Fig. 6B), although this is not
surprising because similar results have been reported by others in different
cell types (Ding et al., 2000
;
Smalley et al., 1999
).
Besides WNTs, GSK3ß is a key component of several signalling pathways.
Inhibition of this constitutive active enzyme involves either formation of
protein complexes, as in response to WNTs, or phophorylation of specific Ser
residues (Cohen and Frame,
2001). Time-course immunoblot analysis with GSK3ß
phosphorylation specific antibodies demonstrated that SFRP1 CM treatment is
followed by a progressive increase in GSK3ß phosphorylation at
Ser9, indicative of its inhibition, whereas its phophorylation at
Tyr216, which is a measure of the active state
(Hughes et al., 1993
), did not
change between control and treated cultures
(Fig. 7A).
|
A possible explanation of this result was that a trophic factor co-purified with Sfrp1 and produced by MDCK cell line in response to Sfrp1 transfection was responsible for GSK3ß Ser9 phosphorylation. To exclude this possibility, E5 retina explants were electroporated either with Gfp or with Sfrp1 and Gfp as a tracer. Explants were grown for 13 hours in defined medium and collected for western blot analysis approximately five hours after protein expression was visually assessed. Without altering the total level of the protein, Sfrp1 overexpression induced a clear increase in GSK3ß Ser9-phosphorylation (Fig. 7B), demonstrating that this modification is likely to be a consequence of Sfrp1 activity.
Together these results suggest that GSK3ß might be an effector of SFRP1 activity in the chick retina.
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Discussion |
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SFRPs have been implicated, as Wnt signalling antagonists, in the control
of axis formation in Xenopus embryos
(Kiecker and Niehrs, 2001),
vertebrate somite development (Borello et
al., 1999
; Lee et al.,
2000a
), regulation of apoptosis
(Ellies et al., 2000
;
Melkonyan et al., 1997
), and
kidney and heart formation (Lescher et
al., 1998
; Schneider and
Mercola, 2001
; Yoshino et al.,
2001
). On the basis of the expression pattern in the embryonic
chick eye, we have addressed here the role of SFRP1 during retina development.
Both gain- and loss-of-function experiments suggested that SFRP1 favoured the
number of differentiated neurons in dispersed retina cell cultures.
RCAS-mediated overexpression of SFRP1 in ovo extended these observations,
demonstrating that SFRP1 favours the generation of both RGC and cone
photoreceptors while decreasing the number of amacrine cells. These changes
did not imply variations in the rate of mitotic cell division or cell
death.
A proposed model for the mechanisms that control retina neurogenesis
suggests that the withdrawal of a retina progenitor cell from the cell cycle
and its commitment to a differentiated neuron or glia phenotype requires the
coordination between a competence state of the precursor cell and its
capability of interpreting different extrinsic signals provided by adjacent
cells (Livesey and Cepko,
2001). In this context, our findings are consistent with the idea
that SFRP1 acts as a differentiation signal on a continuously available pool
of progenitor cells. Increased amount of SFRP1 during the first wave of neuron
generation appears to drive more progenitor cells into the earliest born
retinal cell types, RGC and cones. As a consequence, only a decreased pool of
progenitors is available for the generation of neurons born in the second wave
of retina neurogenesis. In our analysis we have consistently found a decrease
in the number of amacrine cells, which birth date partially overlaps with that
of RGC and cones, and, at later stages, a slight decrease in that of
horizontal cells. Because these losses appear to be insufficient to compensate
for the increase in cones and RGC, it is possible that other cell types might
be affected later on, although we were unable to detect it.
Both extrinsic and intrinsic cues contribute to the determination of retina
cell fates. Similarly to SFRP1, alterations in the proportion of retina cell
types have been observed after manipulations in a number of cues thought to
act on already competent progenitors or on post-mitotic cells `en route' to
differentiation (Cepko, 1999).
These include transcription factors of the bHLH family, such as the
Ath5 or NeuroD genes
(Brown et al., 2001
;
Kay et al., 2001
;
Moore et al., 2002
;
Morrow et al., 1999
;
Wang et al., 2001
), or
secreted signalling molecules such as CNTF or FGFs
(Ezzeddine et al., 1997
;
McFarlane et al., 1998
;
Patel and McFarlane, 2000
).
Our experimental design (overexpression during early neurogenesis) does not
allow us to distinguish whether SFRP1 is a specific selector for RGC and cone
photoreceptors or whether it just pushes more cells toward differentiation
into the scheduled phenotype. The dynamic expression of Sfrp1, which
follows the wave of neurogenesis in the retina, might support the latter
hypothesis. Should this be the case, SFRP1 overexpression at later stages of
neurogenesis would increase, for instance, the number of amacrine/horizontal
cells over bipolar cells.
The activation of the Delta-Notch signalling pathway is one of the key
events in the process of cell differentiation. Although Notch signalling may
be required at different stages of retina development
(Livesey and Cepko, 2001),
interference with its activity at early stages of development in the chick
increased the production of RGC (Austin et
al., 1995
; Henrique et al.,
1997
). Because Notch activation decreases Delta
expression, it has been proposed that waves of Delta expression may
control the numbers of retina progenitors competent to respond to
time-controlled inductive signals, thus allowing the generation of
sequentially ordered cell fates (Dorsky et
al., 1997
). SFRP1 overexpression did not appear to modify the
level of Delta expression in the retina, and ectopic expression of
SFRP1 in the proliferating peripheral retina did not initiate ectopic neuron
production. Altogether these data indicate that only already competent cells
could differentiate upon exposure to SFRP1.
Wnt genes were first described in the CNS
(Wilkinson et al., 1987);
nevertheless, their functions in this tissue are still poorly understood.
Different WNTs have been implicated in the control of neural progenitor
proliferation (Dickinson et al.,
1994
; Ikeya et al.,
1997
; Lee et al.,
2000b
). Recently, it has been proposed that in the neural tube
this function might be achieved through the establishment of a mitogenic
gradient, that, upon activation of the canonical signalling pathway exerts a
transcriptional regulation of cell cycle specific genes, controlling the final
size and shape of the neural tube across the dorso-ventral axis
(Megason and McMahon, 2002
).
On the basis of these observations and on the proposed function for SFRP
proteins, SFRP1-mediated effects observed in the retina could be explained as
the results of SFRP1 interference with endogenously present mitogenic,
ß-catenin-dependent WNTs. Our results, however, do not easily support
this possibility. Comparison of the reporter activity of a wild-type or a
mutated version of the TCF-responsive element suggests that E5 chick retina
cells have a very low basal ß-catenin transcriptional activity. This
activity was not modified by different amounts of Sfrp1, although
Sfrp1 could, at least in part, repress the activation of
ß-catenin transcriptional activity induced by Wnt8 transfection.
These data suggest that retina cells are competent to respond to Wnt
signalling and that SFRP1 has the potential of antagonising WNTs, when these
are exogenously added. However, SFRP1 activity in the retina appears
independent at least of those WNTs that activate the canonical pathway. Theses
results are in agreement with a recent study in zebrafish embryos that shows
that, in the eye, cells from the pigment epithelium, lens and ciliary margin
but not those from the neural retina are potential ß-catenin-responsive
cells (Dorsky et al.,
2002
).
Besides ß-catenin, Wnt signalling can lead either to the activation of
protein kinase C or to Jun-kinase mediated transcription. At the moment we
cannot exclude that SFRP1 activity in the retina interferes with one of these
pathway. However, initial experiment aimed at determining this possibility, do
not support this idea (data not shown). Instead our results indicate that
SFRP1 treatment is followed by a phospho-Ser9 mediated inhibition
of GSK3ß, and RCAS-mediated ectopic expression of a dominant-negative
form of GSK3ß induced a retina phenotype remarkably similar to that
obtained with SFRP1 gain-of-function. Phosphorylation mediated inhibition of
GSK3ß is a central step over which the signalling cascades of several
different trophic factors converge, but this is not the mechanism implied in
GSK3ß inhibition in the canonical Wnt pathway
(Ding et al., 2000). Therefore
our results are, again, not in line with the proposed function for SFRPs. Our
results do not establish a direct link between SFRP1 and GSK3ß
phosphorylation and we cannot totally exclude that RCAS-SFRP1 and
RCAS-dnGSK3ß mediated phenotypes in the retina might be a mere
coincidence. However, frizzled signalling has been proposed as a possible
trigger for similar GSK3ß-mediated phenotypes observed in the
Xenopus retina, where GSK3ß activity seems required for the
post-translational control of different bHLH molecules, needed for the
time-controlled generation of the various retina neurons
(Moore et al., 2002
). Whether
SFRP1 might be part of this trigger is just a matter of speculation. As a
hypothesis, in absence of WNTs molecule, SFRP1 could bind directly to a Fz
receptor through interaction of their respective CRD domains. Indeed, SFRP1
can form complexes with a Fz prototype
(Bafico et al., 1999
), and
dimerization and multimerization of the CRD domains in both Fz and SFRPs
appears as a necessary step for their function
(Dann et al., 2001
). Fz
activation or SFRP1 binding to a different receptor (i.e. a receptor with
intrinsic protein tyrosine kinase activity) could lead to activation of a
kinase that, in turn, would be responsible for the phospho-Ser9-mediated
inactivation of GSK3ß (Cohen and
Frame, 2001
), followed by the post-transcriptional control of
genes, as those related to atonal, required for the acquisition of specific
cell phenotype (Moore et al.,
2002
). This model does not exclude that, in other tissues, in the
presence of WNT, SFRP1 may interfere with the canonical signalling pathway as
an extra-cellular antagonist, as previously described
(Bafico et al., 1999
;
Xu et al., 1998
).
In conclusion, we propose that SFRP1 contributes to retina development with a mechanism that does not involve preventing WNT-mediated activation of its canonical signalling pathway. Whether SFRP1 interferes with non-canonical Wnt signalling still needs to be determined.
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