1 Department of Cell and Developmental Biology, Graduate School of Biostudies,
Kyoto University, Kitashirakawa Oiwake-cho, Kyoto 606-8502
2 RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami-Cho, Kobe
650-0047, Japan
* Author for correspondence at address 2 (e-mail: nakagawa{at}cdb.riken.go.jp)
Accepted 31 October 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Wnt, Frizzled, Canonical pathway, Chicken, Retina, Stem cell, Differentiation, Electroporation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The CMZ progenitor cells are distinct from other progenitor cells in the
central part of the developing retina, although both of them produce all the
retinal neurons and glia (Layer et al.,
2001; Perron and Harris,
2000
; Reh and Levine,
1998
). For example, in Xenopus, the CMZ progenitor cells
produce a large number of progenitor clones forming wide columns, whereas the
retinal progenitor cells in the central retina undergo a limited number of
cell divisions to generate progenies forming narrow radial columns
(Holt et al., 1988
;
Wetts and Fraser, 1988
;
Wetts et al., 1989
). In
addition, the embryonic retinal margin, but not the central retina, possesses
the capacity to regenerate a correctly laminated neural retina after
retinoectomy, despite the fact that both of the retinal regions contain
multipotential retinal progenitor cells
(Coulombre and Coulombre,
1965
; Coulombre and Coulombre,
1970
). The two progenitor populations can also be distinguished by
molecular marker expression. Both of the retinal progenitor cells are
identified by co-expression of Pax6 and Chx10
(Belecky-Adams et al., 1997
;
Fischer and Reh, 2000
). The
CMZ progenitor cells, however, do not express neural precursor marker Notch1
(Dorsky et al., 1995
;
Perron et al., 1998
), which is
strongly expressed in the proliferating progenitor cells in the central retina
(Austin et al., 1995
;
Henrique et al., 1997
). It is
generally accepted that the continuous proliferation of the CMZ progenitor
cells add cells peripherally during the embryonic period, accounting at least
in part for the centroperipheral gradient of neural differentiation
(Coulombre, 1965
;
Layer and Willbold, 1993
).
To address the molecular mechanism that controls the prolonged
proliferation of the CMZ progenitor cells, we employed a candidate molecule
approach. Wnt genes were originally identified as oncogenes, and had been
shown to regulate a variety of developmental processes (reviewed by
Wodarz and Nusse, 1998). We
noted that many of the Wnt family molecules were expressed in the region where
multipotential progenitor cells are maintained, such as the neural crest
cell-producing dorsal neural tube, primitive streak, tail bud and
proliferating region of the hair follicles
(Wodarz and Nusse, 1998
). In
the present study, we analyzed the function of chicken Wnt2b expressed in the
marginal-most tip of the embryonic chicken retina
(Jasoni et al., 1999
). When
ectopically overexpressed in ovo, Wnt2b induced expression of molecular
markers characteristics of CMZ progenitor cell. On the contrary, blocking of
Wnt2b function by a dominant-negative LEF1 inhibited proliferation of the
marginal cells, leading to their premature differentiation. Furthermore, the
CMZ progenitor cells proliferated for a longer period than the centrally
located ones, and their proliferation was further promoted by chicken Wnt2b in
vitro. Our results suggest that Wnt2b regulates the maintenance of a retinal
progenitor population in the CMZ, and thus function as a putative stem cell
factor in the retina.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular cloning and construction of the vectors
Degenerate primers were designed for conserved sequences of Frizzled family
proteins, YPERPII and YFHLAAW, and RT-PCR was carried out by using cDNA from
E5 chicken embryonic retina as a template. The amplified fragments were
subcloned into pCR2.1 (Invitrogen), sequenced, and subsequently used for
making cRNA probes for in situ hybridization. For amplifying Wnt family genes,
the same set of degenerated primers were used as previously described
(Gavin et al., 1990). To
obtain a cDNA fragment of chicken Ptmb4, we carried out RT-PCR by using the
following the following set of primers: 5'-atgtctgacaaaccagatat-3'
and 5'-ttcttacaaagttaaacagc-3'. To obtain a full-length clone of
the mouse Wnt2b gene, we designed primers according to the published sequences
(Katoh et al., 1996
) and
amplified the entire coding region by RT-PCR using cDNA from E14 retinas. The
construction of pcDNA3.1-mFzd4-CRD-myc and hFzd5-CRD-myc was described
elsewhere (S. N., S. Takada, R. Takada and M. T., unpublished). For in ovo
overexpression studies, cDNA fragments were subcloned into pCA-pA
(Niwa et al., 1991
), which
enabled strong exogenous expression in chicken embryos
(Momose et al., 1999
). The
cDNA clone of chicken Wnt2b was a kind gift from Dr Izpisua Belmonte
(Kawakami et al., 2001
). The
entire coding region of chicken Wnt2b was subcloned into pEF-Fc
(Suda and Nagata, 1994
) to
generate a fusion protein of chicken Wnt2b and the Fc region of human IgG.
RCAS-
LEF1 was a kind gift from Dr Kengaku, and the coding region was
subcloned into pCA-pA for the in ovo electroporation study. The GenBank
Accession Number for chicken Frizzled 5 is AF463494.
Fzd-binding study using immunoprecipitation
As both Wnt2b-Fc and the Fzds-CRD-myc were poorly secreted into the culture
medium, we analyzed the binding of those proteins by co-transfecting cells
with both proteins, followed by immunoprecipitation of Wnt2b-Fc. COS7 cells
were cultured in DH10, which is 1:1 mixture of Dulbecco's MEM (DMEM, Nissui,
Japan) and Ham's F12 (F12, Nissui, Japan) supplemented with 10% FCS, 0.3%
glucose and antibiotics. The cells (3x105) were
co-transfected with pcDNA3.1-Fzds-CRD-myc and pEF-Wnt2b-Fc by using the
Effectene reagent (Qiagen) according to the manufacturer's instructions. As a
control experiment, pEF-c-cad7-Fc
(Nakagawa and Takeichi, 1998)
was used instead of pEF-Wnt2b-Fc. After 48 hours, the cells were washed twice
with phosphate-buffered saline (PBS) and incubated in 1 ml of binding buffer
(1% Triton-X-100, 1% NP-40, 1 mM EDTA in PBS) for 30 minutes at 4°C. The
cell lysates were then centrifuged at 17,400 g for 5 minutes
to remove the cell debris and incubated with 50 µl of Protein-A
Sepharose-4B beads (Zymed) for 30 minutes at 4°C. After extensive washing
with the binding buffer, the immunoprecipitated complexes were lysed in
SDS-PAGE sample buffer, run on a 10% acrylamide gel, and then analyzed by
using a standard western blotting technique. For detecting the myc and the Fc
antigen, we used a mouse monoclonal antibody 9E10 (Sigma) and rabbit
anti-human IgG-Fc (Jackson Laboratory), respectively, followed by
HRP-conjugated goat anti-mouse IgG (Amersham) and HRP-conjugated goat
anti-rabbit IgG (Amersham), respectively.
Preparation of conditioned medium of chicken Wnt2b producing cultures
and the lysate of mFzd4-CRD producing cells
To obtain functional chicken Wnt2b proteins, we collected conditioned
medium (CM) prepared from 293 cells stably transfected with pCA-Wnt2b. The
transfectants were grown to confluence, and the medium was replaced with fresh
DH10, and the cells were further incubated for 48 hours. The supernatants were
collected and centrifuged at 48,384 g to remove insoluble
materials, filtered, aliquoted and stored at -80°C until used. For
collecting soluble mFzd4-CRD, 5x105 COS-7 were plated on 5 cm
dish and transfected with pcDNA3.1-mFzd4-CRD-myc. On the next day, the medium
was replaced with fresh DH10, and the cells were cultured for 48 hours. The
cells were then washed with PBS, and incubated for 30 minutes in 0.5 ml of 1%
CHAPS in PBS containing 1 mM EDTA on ice. After the centrifugation, the cell
lysates were extensively dialyzed against PBS, and then against DH, and were
thereafter kept frozen until used. As a control, GFP-expressing COS7 cells
were uses as the source of the cell lysates.
Retinal monolayer culture
The cultures were prepared according to the methods previously described
(Willbold et al., 2000) with
minor modifications. Briefly, the marginal part of E5 neural retinas was
dissected in F12 and treated for 10 minutes at room temperature with 0.1%
crude trypsin (1:50; DIFCO) in saline buffered with HEPES (10 mM, pH 7.4) and
supplemented with 1 mM EDTA. After 2 washes in DH10, DNAseI (Sigma) was added
at a final concentration of 0.001% in DH10, and the retinal fragments were
dissociated into single cells by gentle pipetting with a fire-polished Pasteur
pipette. After low-speed centrifuge at 700 g for 5 minutes,
2x106 cells were resuspended in 2 ml of culture medium and
plated into each well of six-well dishes (Falcon). A 300 µl volume of CM
(prepared from chicken Wnt2b-expressing or parental 293 cells) and 700 µl
of cell lysates (prepared from mFz4-CRD or GFP-expressing cells) were then
added to the medium, and the cells were cultured for 6 hours. For preparing
cytoplasmic and membrane-associated ß-catenin, fractionation was
performed according to the procedures described previously
(Shibamoto et al., 1998
). To
detect the ß-catenin on a western blot, we used a rabbit polyclonal
antibody against ß-catenin (Shibamoto
et al., 1998
) and HRP-conjugated goat anti rabbit Ig (Amersham).
After visualization of the signal by using ECL (Amersham), the intensity of
the signals was analyzed with an Image Master (Amersham Pharmacia Biotech).
For RT-PCR to study the gene expression, the following primers were used:
5'-catgtgaagcctcagcac-3' and 5'-cctggataaagctgcatg-3'
for LEF1 and 5'-aagcccattgactttgag-3' and
5'-tggactctcattcacatc-3' for N-cadherin. The PCR conditions used
were 94°C for 45 seconds, 50°C for 45 seconds, and 72°C for 3
minutes, for a total of 25 cycles. The PCR products were electrophoresed on a
2% agarose gel, stained with ethidium bromide, and analyzed with the Image
Master. The amplified fragments were also subcloned into pCR2.1 and sequenced
to confirm specific amplification of the desired genes.
Reaggregation culture for clonal analysis
The reaggregation culture was prepared according to the methods previously
described (Belliveau and Cepko,
1999) with minor modifications. We employed this culture system
because retinal cells plated on a culture dish firmly adhered to the substrate
within 12 hours of the addition of Wnt2b CM, which hampered the analysis
because cell-substrate interaction promoted nonspecific cell differentiation.
Small tissue explants were prepared from the CMZ or the central part of the
neural retina of E4.5 quail retina, treated with 0.1% trypsin and dissociated
into single cells as described above. They were then mixed with an excess of
dissociated cells (4x103-fold) prepared from the central part
of E5 chicken retina, and centrifuged at 700 g for 1 minute. The cell
pellet was then suspended in culture medium at a concentration of
2x105 cells/µl. Cell suspension (1 µl) was placed on a
Millicell CM filter, 0.4 µm pore size (Millipore) and cultured for 7 days.
The culture medium used was DH10 supplemented with control or chick Wnt2b CM,
which was added to the culture medium at a dilution of 1:5. The half volume of
the culture medium was exchanged every 2 days. To examine the secondary clone
formation, we cultured the primary clones for 4 days either in the presence or
absence of chicken Wnt2b CM. The pellets were dissociated into single cells
and then pelleted to be cultured for a further 7 days as described above.
In ovo electroporation
The purified DNA for each plasmid was diluted to a final concentration of 5
µg/µl and mixed with 1.5 µg/µl of the GFP-expressing plasmid to
identify regions of gene introduction. pCA-mWnt2b-pA was used at a
concentration of 1 µg/µl. Fast Green (1%) was added to facilitate
visualization of the injected DNA solution. DNA solution (30 nl) was
injected into the optic vesicles of stage 10-11 embryos. After the injection,
the anode was placed beside the optic vesicle, and a tungsten microelectrode
was inserted into the vesicle through the anterior neuropore. An electric
pulse was applied for 25 ms, three times at 7 V. Electroporated embryos were
incubated at 38.5°C until they reached the appropriate stages. Strong
exogenous expression began approximately 12 hours after electroporation (12
hours AE), corresponding to stage 16-18
(Momose et al., 1999
).
Immunostaining and in situ hybridization
Mouse anti collagen type IX (clone 2C2, Developmental Studies of Hybridoma
Bank; DSHB), mouse anti-Hu (clone 16A11, Molecular Probe), mouse anti-Islet1
(clone 40.2D6, DSHB), mouse anti-glutamine synthetase (clone 6, Transduction),
mouse anti-middle molecular weight neurofilament (clone RMO270, Zymed), mouse
anti-quail nucleus (clone QCPN, DSHB), mouse anti-BrdU (clone BU33, SIGMA),
rabbit anti-visinin (kind gift from Dr Miki)
(Hatakenaka et al., 1985),
rabbit anti-GFP (Chemicon), Cy3-conjugated anti-mouse IgG (Chemicon), Alexa
488-conjugated anti-rabbit IgG (Molecular Probes) and Alexa 647-conjugated
anti-mouse IgG (Molecular Probes) antibodies were used. To study cell
proliferation at E3.5, a few microliters of 50 mM BrdU were injected into an
allantoic vein
1 hour before fixation. For immunostaining experiments,
embryos were fixed for 1 hour at room temperature in 4% paraformaldehyde in
PBS, cryoprotected in 30% sucrose in PBS for 30 minutes, and then embedded in
Tissue-Tek (Sakura). Sections at a thickness of 10 µm were collected on
silane-coated slide glasses, rehydrated in PBS, and permeabilized in 100%
methanol for 5 minutes at -20°C. Following re-hydration in PBS,
nonspecific binding on these samples was blocked with 4% skim milk (Difco) for
5 minutes. For triple-color immunostaining of the reaggregation culture, the
pellets were fixed in 4% paraformaldehyde for 1 hour at room temperature,
permeabilized in -20°C methanol for 15 minutes. After dehydration with
PBS, nonspecific binding was blocked with PBST (0.2% Triton X-100 in PBS)
supplemented with 5% fetal bovine serum (FBS), and the reaggregates were
incubated with primary antibodies overnight at 4°C. After extensive
washing with PBT, they were incubated with secondary antibodies for 4-5 hours.
After extensive washing, they were then incubated with a second set of primary
antibodies that had been directly labeled with either ZENON Alexa 555
(Molecular Probe; for labeling QCPN) or Alexa-488 Antibody labeling kit
(Molecular Probe; for labeling HuD). Confocal images at a thickness of 1.7
µm optical sections were collected using LSM510 (Zeiss). For in situ
hybridization studies, embryos were fixed in 4% paraformaldehyde overnight at
4°C and processed using standard protocols
(Perron et al., 1998
). The
cRNA probes for LEF1 and Notch1 were kindly provided by Dr Kengaku (Kyoto
University, Japan) and Dr Wakamatsu (Tohoku University, Japan), respectively,
and those for Chx10, Pax6 and Rx1 by Dr Sakagami and Dr Yasuda (Nara Institute
for Science and Technology, Japan). For the Wnt, Frizzled and Ptmb4 sequences,
the PCR fragments described above, cloned into pCR2.1, were used as
templates.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Wnt proteins transmit their signal by binding to a member of the Frizzled
family molecules, which are seven-pass transmembrane receptors
(Wodarz and Nusse, 1998). To
characterize the subtypes of Frizzled proteins that would mediate the chicken
Wnt2b signaling from the eye margin, we carried out RT-PCR again, using
primers designed for conserved sequences of the Frizzled transmembrane
domains. We obtained cDNA fragments that were identical to previously cloned
chicken Frizzled 3 and Frizzled 4 (Nohno
et al., 1999
), in addition to one fragment that showed its highest
sequence similarity to human Frizzled 5
(Wang et al., 1996
). We then
examined the expression pattern of these chicken Frizzled proteins (Fzd3, 4,
5) at stage 22 by in situ hybridization. The Fzd4 and Fzd5 mRNAs were
expressed in the peripheral (Fig.
1B) and the central (Fig.
1C) region of the retina, respectively. We could not detect
specific signals for Fzd3 in the retina at the stage tested (data not
shown).
We subsequently examined the expression pattern of LEF1, which is an
essential component in one of the Wnt signaling pathways called the canonical
pathway and forms a transcriptional activator complex with ß-catenin
(Behrens et al., 1996;
Molenaar et al., 1996
). In the
chicken embryo, LEF1 mRNA is highly expressed where the Wnt-canonical pathway
is operating, such as the apical ectodermal ridge of developing limb buds,
medial somites, primitive streak and tail buds
(Kengaku et al., 1998
;
Schmidt et al., 2000
). In
addition, exogenous activation of the Wnt signaling pathway leads to an
upregulation of LEF1 mRNA (Kengaku et al.,
1998
; Schmidt et al.,
2000
). Accordingly, the strong LEF1 mRNA expression is considered
to be a tentative molecular marker for an active Wnt canonical pathway
(Kengaku et al., 1998
;
Schmidt et al., 2000
). The
expression of LEF1 mRNA was particularly higher in the peripheral part of the
retina next to the chicken Wnt2b-expressing region, in which expression
decreased gradually towards the central retina
(Fig. 1D).
To test a possible interaction between chicken Wnt2b and Fzd4 or Fzd5, we
carried out immunoprecipitation experiments using tagged proteins. We
co-transfected COS7 cells with myc-tagged Fzd-CRDs (cysteine-rich domain) and
chicken Wnt2b fused to the Fc region of human IgG. We then prepared cell
lysates and immunoprecipitated the chicken Wnt2b-Fc with Protein-A beads. As a
control experiment, we co-transfected the cells with the extracellular domain
of cadherin 7 fused to the IgG-Fc
(Nakagawa and Takeichi, 1998).
For these experiments, we used mouse frizzled 4 and human frizzled 5
(Wang et al., 1996
), because
the full-length chicken clones were not available at the time of the study.
Both mouse Fzd4-CRD-myc and human Fzd-5-CRD-myc were specifically
co-immunoprecipitated with chicken Wnt2b-Fc, but not with the control cadherin
7-Fc (Fig. 1E), suggesting that
chicken Fzd4 and Fzd5 were able to transmit the chicken Wnt2b signal in the
retina.
To confirm the activation of the Wnt canonical pathway by chicken Wnt2b, we
prepared dissociated cells from E5 retina and cultured them in the presence or
absence of chicken Wnt2b-conditioned medium. We then studied the level of
cytosolic ß-catenin by western blotting, as cytosolic ß-catenin is
stabilized after Wnt canonical pathway activation
(Behrens et al., 1996;
Molenaar et al., 1996
). In
control cultures, little or no cytosolic ß-catenin was detected; however,
the ratio of the cytosolic ß-catenin to the membrane-bound form increased
sevenfold as early as 6 hours after addition of chicken Wnt2b CM
(Fig. 1F,G). We also examined
the upregulation of LEF1 mRNA by chickenWnt2b by using RT-PCR. The LEF1
expression level increased more than fivefold by the addition of chicken Wnt2b
CM, whereas control N-cadherin expression remained constant
(Fig. 1F,G). To confirm that
these effects were specific to the Wnt2b in the conditioned medium, we added a
soluble form of the extracellular domain of Frizzled CRD domain into the
culture. The soluble Frz4-CRD neutralized cytosolic ß-catenin
stabilization and LEF1 mRNA upregulation, indicating the specificity of the
chicken Wnt2b CM for the Wnt pathway (Fig.
1F,G).
LEF1 mRNA is upregulated in a region between the presumptive iris and
the neural retina
The marginal retina is largely divided into three parts: the marginal-most
area forming iris and ciliary epithelium; the centrally located area
determined to become the neural retina; and the intermediate region CMZ,
containing retinal progenitor cells
(Coulombre, 1965;
Perron and Harris, 2000
). To
further characterize the LEF1 mRNA-expressing region, we examined the
expression pattern of various molecular markers using adjacent sections of E5
retina, when the future iris and ciliary epithelium can be morphologically
identified (Bard and Ross,
1982
). At this stage, chicken Wnt2b remained expressed in the
marginal-most tip of the retina (small arrowhead in
Fig. 2A). LEF1 mRNA was
downregulated in the neighboring region (small arrowhead in
Fig. 2E), which strongly
expressed collagen type IX and Ptmb4 (small arrowheads in
Fig. 2C,D), markers for the
presumptive iris or ciliary epithelium
(Thut et al., 2001
). The
marginal region expressed neither Chx10 nor Rx1 (small arrowheads in
Fig. 2F,G), markers for the
retinal progenitor cells (Belecky-Adams et
al., 1997
; Fischer and Reh,
2000
; Ohuchi et al.,
1999
). We could not detect the expression of Notch1 or middle
molecular weight neurofilament (NF-M) in the marginal region, either (small
arrowheads in Fig. 2H,I), which
are markers for the neural precursor cells and the retinal ganglion cells,
respectively (Austin et al.,
1995
; Henrique et al.,
1997
; McCabe et al.,
1999
). Notch1 was highly expressed in the centrally located neural
retina, which also expressed NF-M (large arrowheads in
Fig. 2H,I). Strong LEF1 mRNA
expression was observed in the CMZ, a region between the presumptive
iris/ciliary epithelium and the neural retina (arrow in
Fig. 2E). Weak expression of
Ptmb4 and collagen type IX were also observed in the LEF1-expressing region;
however, their signals were much weaker compared with the signals in the iris
and ciliary epithelium (arrows in Fig.
2C,D). The LEF1 mRNA expressing region also expressed moderate
level of Pax6, Chx10 and Rx1 (arrows in
Fig. 2B,F,G), but did not
express Notch1 or NF-M (arrows in Fig.
2H,I). These observations are consistent with the idea that LEF1
mRNA was upregulated in the CMZ progenitor cells, although we could not
confirm the marker expression at a single cell level because of the diffuse
signals of in situ hybridization.
|
Wnt2b overexpression inhibits expression of differentiation markers
in ovo
To investigate the involvement of chicken Wnt2b in the maintenance of CMZ
progenitor cells, we carried out overexpression experiments using the in ovo
electroporation technique (Momose et al.,
1999). We initially studied the expression pattern of Wnt
signaling components at stage 18, as we examined the effects of the chick
Wnt2b overexpression around this stage (see below). Chicken Wnt2b was
upregulated in the dorsal margin of the invaginating eye bud
(Fig. 3A), and Fzd4 and Fzd5
were expressed in the peripheral and the central region of the retina,
respectively (Fig. 3B,C). The
chicken Wnt2b expression was detected in the ventral margin, as well as dorsal
margin at slightly later stages, such as stage 19 (data not shown). LEF1 was
expressed at high levels in the marginal region, decreasing centrally
(Fig. 3D). We then
electroporated stage 10 embryos with mouse Wnt2b and studied the time course
of the overexpressed message by using a mouse cRNA probe, which did not
crossreact with the endogenous chicken Wnt2b mRNA
(Fig. 3E-G). The ectopic
expression of mouse Wnt2b was first observed in the entire eye bud in addition
to the ventral diencephalon at stage 13 (12 hours after the electroporation,
Fig. 3E). The overexpressed
message was also detected at stage 16 (24 hours after the electroporation,
Fig. 3F), but little or no
signal was observed at stage 21 (48 hours after the electroporation,
Fig. 3G). We then studied LEF1
mRNA expression using the adjacent sections to examine where the Wnt canonical
pathway was activated. We could not detect any LEF1 mRNA at stage 13; however,
it was strongly upregulated at stage 16
(Fig. 3I), whereas little or no
expression was observed in the control retina at that stage (data not shown).
The LEF1 mRNA continued to be expressed ectopically in the central retina at
stage 21, even though the exogenous expression of mouse Wnt2b had decreased
under the detection level (Fig.
3J). Interestingly, the retina electroporated with mouse Wnt2b
became folded at high frequency (Fig.
3G,J). To investigate cellular differentiation in the folded
retina, we studied the expression pattern of molecular markers described
previously (Fig. 4). In the
following experiments, we co-electroporated with a GFP-expressing plasmid to
identify the region where the overexpressed gene was introduced. In control
embryos injected with GFP only, the central part of the retina started to
express neural precursor marker Notch1 and ganglion cell marker NF-M
(Fig. 4A,C). In the folded
retinas expressing mouse Wnt2b, however, neither Notch1 nor NF-M signal was
detected (Fig. 4B,D); no change
was observed in the expression of these markers in the ventral diencephalon of
the same embryo, despite the expression of the co-electroporated GFP
(Fig. 4B,D), suggesting the
specific effect in the retina. We then studied the expression pattern of
progenitor cell markers in the folded retina
(Fig. 4E-J). At this stage, a
strong Pax6 signal was observed only in the marginal area
(Fig. 4E). However, strong
expression of Pax6 was induced in the central part of the mouse
Wnt2b-electroporated retina (Fig.
4F). The cells in the folded retina also expressed other
progenitor markers, i.e. Chx 10 and Rx1
(Fig. 4H,J). We also observed
weak expression of collagen type IX in the central retina of mouse
Wnt2b-electroporated embryo (Fig.
4L). The signals, however, were much weaker compared with those in
the E5 iris or ciliary epithelium (Fig.
2C). These finding suggests that the cells in the folded retina
were equivalent to the CMZ progenitor cells. Notably, these effects were not
restricted to the cells expressing co-electroporated GFP, and whole retina was
equally affected even when chicken Wnt2b was introduced in a mosaic pattern
(Fig. 4). We also studied the
incorporation of BrdU in the chicken Wnt2b-electroporated embryo to see if
cell proliferation is promoted. At this stage, essentially all the cells
incorporated BrdU and we could not detect any obvious change in the intensity
of the BrdU signals or number of cells positive for it (data not shown).
|
|
Functional blocking of Wnt2b leads to premature ganglion cell
differentiation
We then carried out complementary experiments to block the chicken Wnt2b
downstream signaling by overexpressing a dominant-negative form of LEF1
(LEF1) (Kengaku et al.,
1998
) by using the same in ovo electroporation technique. At stage
21,
LEF1-expressing cells tended to form small cell clumps in the
dorsal retinal margin, which expressed stronger co-electroporated GFP compared
with the control embryo (Fig.
5B,D). Because the cell division was assumed to dilute the number
of plasmids in each cells, the strong GFP signals suggested the cell
proliferation had been inhibited by
LEF1 overexpression. This decrease
in cell proliferation was confirmed by the reduced BrdU incorporation by the
LEF1-expressing cells (Fig.
5G,H). The cells in the clumps expressed NF-M
(Fig. 5K,L), suggesting that
the marginal cells had prematurely differentiated into retinal ganglion cells.
We also studied the expression of other post-mitotic neuronal markers such as
Islet1 (Fig. 5N,P) and Hu (data
not shown) in the
LEF1-electroporated embryos. These markers, however,
were not expressed in the marginal cells, even when
LEF1 had been
introduced by electroporation (Fig.
5P).
|
The multipotential progenitor cells in the CMZ proliferate over a
longer period than the centrally located progenitor cells
Finally, we carried out clonal analysis to examine the proliferation and
differentiation capacity of the CMZ progenitor cells that expressed a high
level of LEF1 mRNA, and compared these cells with progenitor cells located in
the central retina, which expressed little or no LEF1 mRNA. We employed the
reaggregate culture system previously described
(Belliveau and Cepko, 1999)
because the progenitor cells did not grow very well at a clonal density in a
monolayer culture. We first prepared singly dissociated cells from either the
CMZ or central retina of E4.5 quail embryo (equivalent to E5 in chicken
embryo). The dissection of the CMZ was carefully carried out to exclude any
contaminating cells from the iris or neural retina, which was confirmed by the
marker expression in the dissected CMZ explant (data not shown). The
dissociated cells were then mixed with an excess amount of feeder cells
prepared from E5 chicken retinas to dilute the quail cells at a clonal
density, pelleted on a filter and cultured for a further 7 days. In the
reaggregation culture,
70% of the dissociated cells proliferated to make
clones regardless of the region from which they had been prepared, suggesting
the existence of progenitor cells in each region of the retina
(Fig. 6A,B;
Fig. 7A,E). The CMZ progenitor
cells, however, produced a larger number of progenies (average=18.20;
Fig. 6B, Fig. 7E) compared with the
progenitor cells in the central neural retina (average=4.58;
Fig. 6A,
Fig. 7A). We subsequently
examined the expression of differentiation markers in these clones to study if
the clones were multipotential. The markers we used were visinin
(photoreceptor cells), Hu (ganglion cells and amacrine cells) and glutamine
synthetase (Müller glia). At least two of these differentiation markers
were typically expressed within a clone derived from a progenitor cell taken
either from the central retina (Fig.
7B-D) or the CMZ (Fig.
7F-H), suggesting that both progenitor cells were multipotential.
To study the effect of chicken Wnt2b on the differentiation and proliferation
of the progenitor cells, we cultured them in the presence of chicken Wnt2b CM
and examined the number of progenies and the expression of the differentiation
markers. The number of progenitor clones was significantly increased by the
addition of chicken Wnt2b CM (Fig.
6C,D; Fig. 7I,M),
suggesting proliferation of the progenitor cells was promoted in the presence
of chicken Wnt2b, regardless of the retinal region from which they were
prepared. The CMZ clones, however, generally contained larger numbers of
progenies (average=84.04; Fig.
6D, Fig. 7M) than
the clones that had originated from the central progenitor cells
(average=17.51; Fig. 6B,
Fig. 7I). These clones
contained both neurons and glia, suggesting that cellular differentiation had
occurred normally even in the continuous presence of chicken Wnt2b CM
(Fig. 7J-L,N-P). To test if
cell proliferation continued for a longer period in the presence of chicken
Wnt2b, we dissociated the primary clones into single cells after 4 days in
culture, and cultured them once again in the pellets to make secondary clones.
All the cells remained as a single cells in the secondary reaggregation
culture, when they had been cultured in the control CM to make the primary
clones (data not shown), suggesting that all of the progenitor cells had
became postmitotic under that culture condition. However,
10% of the
primary progenies derived from the CMZ progenitor cells made secondary clones
if they had been cultured in the presence of chicken Wnt2b CM during the first
4 days (Fig. 7Q). These
secondary clones contained cells expressing visinin, Hu and glutamine
synthetase, suggesting that they were multipotential
(Fig. 7R-T). The progenitor
cells prepared from the central retina, however, remained as single cells even
if they had been cultured in the presence of chicken Wnt2b (data not
shown).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The marginal-most region of the retina differentiates into non-neural
anterior eye structures such as the iris and ciliary epithelium
(Coulombre, 1965). Because the
chicken Wnt2b-expressing region corresponds to the future rim of the iris, the
inhibition of neuronal differentiation by Wnt2b overexpression could possibly
be explained by transformation of the central neural retina into the
non-neural eye structures. This may apparently agree with our observation that
the expression of collagen type IX was induced in the central part of the
folded retina in the chicken Wnt2b-electroporated embryos. However, the
collagen type IX signals in the folded retina were much weaker than those in
the presumptive iris and ciliary epithelium of the E5 retina, when the first
morphological transformation for their differentiation is observed
(Bard and Ross, 1982
). In
addition, the folded retina expressed retinal progenitor markers such as Chx10
and Rx1, which were not expressed in the retinal region forming the iris and
ciliary epithelium. Considering that the weak expression of collagen type IX
was also observed in the CMZ of the E5 retina, Wnt2b supposedly inhibits both
neuronal and iris/ciliary epithelium differentiation by maintaining the
progenitor cell state, rather than by transforming the neural retina into
non-neural tissues.
The upregulation of LEF1 mRNA is considered to be a tentative marker for the active state of the Wnt downstream pathway. Indeed, our RT-PCR analysis showed that LEF1 mRNA was upregulated in vitro upon the addition of chicken Wnt2b CM to the retinal cell culture. The upregulation of LEF1 mRNA was also induced when chicken Wnt2b was overexpressed by the electroporation in vivo. In the developing retina, the LEF1-expressing region was not restricted to the cells immediately next to the chicken Wnt2b expressing retinal margin, but was widely distributed making a gradient decreasing centrally. In Drosophila imaginal disks, Wingless proteins are transported for a long distance by the argosome (Greco, 2001). It is possible that a similar mechanism also works in the vertebrate neural retina to activate the Wnt signaling pathway at a distance. The long-range effect of Wnt is supported by our observations that the cellular differentiation was inhibited by chicken Wnt2b in a region not neighboring the cells expressing co-electroporated GFP and the whole retina was uniformly affected by relatively uneven introduction of the gene. Alternatively, it is equally possible that LEF1 mRNA is kept upregulated for a while, even when the Wnt ligand is no longer available, and the LEF1-expressing cells are simply displaced centrally by addition of new cells at the peripheral region. Direct observation of chicken Wnt2b protein is crucial to discriminate these two possibilities.
At E5, we observed downregulation of LEF1 mRNA in the presumptive iris and
ciliary epithelium immediately next to the chicken Wnt2b-expressing marginal
tip. It should be noted that the morphological change caused by the iris
differentiation begins at E5 (Bard and
Ross, 1982), which correlates with the downregulation of LEF1 mRNA
in the corresponding regions. The identities of the non-neural marginal retina
are determined through interaction between the retina and the neighboring
lens, which produces diffusible factors that regulate the formation of the
anterior eye structures (Breitman et al.,
1989
; Stroeva,
1960
). Considering that the lens-derived factors were shown to act
over a short range (Thut et al.,
2001
), the factors may inhibit Wnt downstream signaling in a
region close to the lens to induce iris and ciliary epithelium
differentiation. The induction of iris and ciliary epithelium by the lens
factor may also account for the absence of Islet1 and Hu expression in the
marginal retina of the
LEF1-electroporated embryo, which prematurely
expressed NF-M. In the chicken embryonic retina, the expression of NF-M starts
much earlier than that of other ganglion cell markers
(McCabe et al., 1999
). The
lens factor may thus inhibit the maturation of the ectopic ganglion cells
expressing NF-M into Islet1- or Hu-positive ganglion cells in the vicinity of
the lens.
While the expression of the neuronal markers was completely inhibited by in
ovo overexpression of chicken Wnt2b, neuronal differentiation occurred
normally in the reaggregation cultures, even in the continuous presence of
chicken Wnt2b CM. As Wnt proteins are poorly secreted into the medium
(Wodarz and Nusse, 1998), the
amount of the protein in the conditioned medium may not be enough for complete
inhibition of their neuronal differentiation. Alternatively, additional
factors are required to keep CMZ progenitor cells undifferentiated, which are
not present in the feeder cells prepared from the central part of the E5
neural retina. Considering that the retinal cell differentiation is regulated
by both intrinsic and extrinsic factors
(Belliveau and Cepko, 1999
),
progenitor cells may require factors present in the CMZ in addition to chicken
Wnt2b to keep them undifferentiated. This idea could be tested by using CMZ
cells as feeder cells to make the reaggregation pellets; however, this is not
feasible because of the small size of the tissue for collection of the feeder
cells.
Chicken Wnt2b was expressed in the marginal-most tip, and chicken Fzd4 and
Fzd5 were expressed in the marginal and the central part of the retina,
respectively. Although both Fzd proteins bind to chicken Wnt2b in vitro,
chicken Fzd4 would supposedly play a major role for transmitting the
endogenous chicken Wnt2b signal from the eye margin considering its spatial
distribution; and a smaller amount of chicken Wnt2b would thus be available in
the central region expressing a high level of chicken Fzd5. Although we could
not detect the expression of other Frizzled genes in the embryonic chicken
retina, a previously conducted RNase protection assay showed that many of the
mouse frizzled genes (Fzd2-7) were expressed in the adult mouse eye
(Wang et al., 1996). In
addition, frizzled 3 is expressed in the central nervous system, including the
presumptive eye field, in Xenopus, and overexpression of this gene
led to ectopic eye formation (Rasmussen et
al., 2001
). It is therefore possible that these frizzled genes are
also expressed in the chicken retina and transmit the Wnt signals to regulate
multiple aspects of retinal development. It should also be noted that the
soluble forms of the frizzled-related proteins Sfrp2 and Sfrp5 are expressed
in the neural retina and retinal pigment epithelium, respectively
(Chang et al., 1999
),
suggesting that Wnt signaling is modulated in a region-specific or
stage-specific manner. Further studies will definitely be required for the
complete understanding of Wnt function during ocular development.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmad, I., Tang, L. and Pham, H. (2000). Identification of neural progenitors in the adult mammalian eye. Biochem. Biophys. Res. Commun. 270,517 -521.[CrossRef][Medline]
Austin, C. P., Feldman, D. E., Ida, J. A., Jr and Cepko, C.
L. (1995). Vertebrate retinal ganglion cells are selected
from competent progenitors by the action of Notch.
Development 121,3637
-3650.
Bard, J. B. and Ross, A. S. (1982). The morphogenesis of the ciliary body of the avian eye. II. Differential enlargement causes an epithelium to form radial folds. Dev. Biol. 92,87 -96.[Medline]
Beach, D. H. and Jacobson, M. (1979). Patterns of cell proliferation in the retina of the clawed frog during development. J. Comp. Neurol. 183,603 -613.[Medline]
Behrens, J., von Kries, J. P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R. and Birchmeier, W. (1996). Functional interaction of ß-catenin with the transcription factor LEF-1. Nature 382,638 -642.[CrossRef][Medline]
Belecky-Adams, T., Tomarev, S., Li, H. S., Ploder, L., McInnes, R. R., Sundin, O. and Adler, R. (1997). Pax-6, Prox 1, and Chx10 homeobox gene expression correlates with phenotypic fate of retinal precursor cells. Invest. Ophthalmol. Vis. Sci. 38,1293 -1303.[Abstract]
Belliveau, M. J. and Cepko, C. L. (1999).
Extrinsic and intrinsic factors control the genesis of amacrine and cone cells
in the rat retina. Development
126,555
-566.
Breitman, M. L., Bryce, D. M., Giddens, E., Clapoff, S., Goring, D., Tsui, L. C., Klintworth, G. K. and Bernstein, A. (1989). Analysis of lens cell fate and eye morphogenesis in transgenic mice ablated for cells of the lens lineage. Development 106,457 -463.[Abstract]
Chang, J. T., Esumi, N., Moore, K., Li, Y., Zhang, S., Chew, C.,
Goodman, B., Rattner, A., Moody, S., Stetten, G. et al.
(1999). Cloning and characterization of a secreted
frizzled-related protein that is expressed by the retinal pigment epithelium.
Hum. Mol. Genet. 8,575
-583.
Coulombre, A. J. (1965). Organogenesis. New York: Academic Press.
Coulombre, J. L. and Coulombre, A. J. (1965). Regeneration of neural retina from the pigmented epithelium in the chick embryo. Dev. Biol. 12,79 -92.[Medline]
Coulombre, J. L. and Coulombre, A. J. (1970). Influence of mouse neural retina on regeneration of chick neural retina from chick embryonic pigmented epithelium. Nature 228,559 -560.[Medline]
Dorsky, R. I., Rapaport, D. H. and Harris, W. A. (1995). Xotch inhibits cell differentiation in the Xenopus retina. Neuron 14,487 -496.[Medline]
Fischer, A. J. and Reh, T. A. (2000). Identification of a proliferating marginal zone of retinal progenitors in postnatal chickens. Dev. Biol. 220,197 -210.[CrossRef][Medline]
Gavin, B. J., McMahon, J. A. and McMahon, A. P. (1990). Expression of multiple novel Wnt-1/int-1-related genes during fetal and adult mouse development. Genes Dev. 4,2319 -2332.[Abstract]
Greco, V., Hannus, M. and Eaton, S. (2001). Argosomes: a potential vehicle for the spread of morphogens through epithelia. Cell 106,633 -645.[Medline]
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49 -92.
Hatakenaka, S., Kiyama, H., Tohyama, M. and Miki, N. (1985). Immunohistochemical localization of chick retinal 24 kD protein (visinin) in various vertebrate retinae. Brain Res. 331,209 -215.[CrossRef][Medline]
Henrique, D., Hirsinger, E., Adam, J., Le Roux, I., Pourquie, O., Ish-Horowicz, D. and Lewis, J. (1997). Maintenance of neuroepithelial progenitor cells by Delta-Notch signalling in the embryonic chick retina. Curr. Biol. 7, 661-670.[Medline]
Hollyfield, J. G. (1968). Differential addition of cells to the retina in Rana pipiens tadpoles. Dev. Biol. 18,163 -179.[Medline]
Holt, C. E., Bertsch, T. W., Ellis, H. M. and Harris, W. A. (1988). Cellular determination in the Xenopus retina is independent of lineage and birth date. Neuron 1, 15-26.[Medline]
Jasoni, C., Hendrickson, A. and Roelink, H. (1999). Analysis of chicken Wnt-13 expression demonstrates coincidence with cell division in the developing eye and is consistent with a role in induction. Dev. Dyn. 215,215 -224.[CrossRef][Medline]
Johns, P. R. (1977). Growth of the adult goldfish eye. III. Source of the new retinal cells. J. Comp. Neurol. 176,343 -357.[Medline]
Katoh, M., Hirai, M., Sugimura, T. and Terada, M. (1996). Cloning, expression and chromosomal localization of Wnt-13, a novel member of the Wnt gene family. Oncogene 13,873 -876.[Medline]
Kawakami, Y., Wada, N., Nishimatsu, S. I., Ishikawa, T., Noji, S. and Nohno, T. (1999). Involvement of Wnt-5a in chondrogenic pattern formation in the chick limb bud. Dev. Growth Differ. 41,29 -40.[CrossRef][Medline]
Kawakami, Y., Wada, N., Nishimatsu, S. and Nohno, T. (2000). Involvement of frizzled-10 in Wnt-7a signaling during chick limb development. Dev. Growth Differ. 42,561 -569.[CrossRef][Medline]
Kawakami, Y., Capdevila, J., Buscher, D., Itoh, T., Rodriguez Esteban, C. and Izpisua Belmonte, J. C. (2001). WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo. Cell 104,891 -900.[CrossRef][Medline]
Kengaku, M., Capdevila, J., Rodriguez-Esteban, C., De La Pena,
J., Johnson, R. L., Belmonte, J. C. and Tabin, C. J. (1998).
Distinct WNT pathways regulating AER formation and dorsoventral polarity in
the chick limb bud. Science
280,1274
-1277.
Layer, P. G., Rothermel, A. and Willbold, E. (2001). From stem cells towards neural layers: a lesson from re-aggregated embryonic retinal cells. Neuroreport 12,A39 -A46.[Medline]
Layer, P. G. and Willbold, E. (1993). Histogenesis of the avian retina in reaggregation culture: from dissociated cells to laminar neuronal networks. Int. Rev. Cytol. 146, 1-47.[Medline]
McCabe, K. L., Gunther, E. C. and Reh, T. A.
(1999). The development of the pattern of retinal ganglion cells
in the chick retina: mechanisms that control differentiation.
Development 126,5713
-5724.
Meyer, R. L. (1978). Evidence from thymidine labeling for continuing growth of retina and tectum in juvenile goldfish. Exp. Neurol. 59,99 -111.[Medline]
Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destree, O. and Clevers, H. (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86,391 -399.[Medline]
Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K. and Yasuda, K. (1999). Efficient targeting of gene expression in chick embryos by microelectroporation. Dev. Growth Differ. 41,335 -344.[CrossRef][Medline]
Nakagawa, S. and Takeichi, M. (1998). Neural
crest emigration from the neural tube depends on regulated cadherin
expression. Development
125,2963
-2971.
Niwa, H., Yamamura, K. and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108,193 -199.[CrossRef][Medline]
Nohno, T., Kawakami, Y., Wada, N., Komaguchi, C. and Nishimatsu, S. (1999). Differential expression of the frizzled family involved in Wnt signaling during chick limb development. Cell. Mol. Biol. 45,653 -659.
Ohuchi, H., Tomonari, S., Itoh, H., Mikawa, T. and Noji, S. (1999). Identification of chick rax/rx genes with overlapping patterns of expression during early eye and brain development. Mech. Dev. 85,193 -195.[CrossRef][Medline]
Perron, M. and Harris, W. A. (2000). Retinal stem cells in vertebrates. BioEssays 22,685 -688.[CrossRef][Medline]
Perron, M., Kanekar, S., Vetter, M. L. and Harris, W. A. (1998). The genetic sequence of retinal development in the ciliary margin of the Xenopus eye. Dev. Biol. 199,185 -200.[CrossRef][Medline]
Rasmussen, J. T., Deardorff, M. A., Tan, C., Rao, M. S., Klein,
P. S. and Vetter, M. L. (2001). Regulation of eye development
by frizzled signaling in Xenopus. Proc. Natl. Acad. Sci.
USA 98,3861
-3866.
Reh, T. A. and Levine, E. M. (1998). Multipotential stem cells and progenitors in the vertebrate retina. J. Neurobiol. 36,206 -220.[CrossRef][Medline]
Schmidt, M., Tanaka, M. and Munsterberg, A.
(2000). Expression of ß-catenin in the developing chick
myotome is regulated by myogenic signals. Development
127,4105
-4113.
Shibamoto, S., Higano, K., Takada, R., Ito, F., Takeichi, M. and
Takada, S. (1998). Cytoskeletal reorganization by soluble
Wnt-3a protein signalling. Genes Cells
3, 659-670.
Straznicky, K. and Gaze, R. M. (1971). The growth of the retina in Xenopus laevis: an autoradiographic study. J. Embryol. Exp. Morphol. 26, 67-79.[Medline]
Stroeva, O. G. (1960). Experimental analysis of the eye morphogenesis in mammals. J. Embryol. Exp. Morphol. 8,349 -368.
Suda, T. and Nagata, S. (1994). Purification and characterization of the Fasligand that induces apoptosis. J. Exp. Med. 179,873 -879.[Abstract]
Thut, C. J., Rountree, R. B., Hwa, M. and Kingsley, D. M. (2001). A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens. Dev. Biol. 231,63 -76.[CrossRef][Medline]
Tropepe, V., Coles, B. L., Chiasson, B. J., Horsford, D. J.,
Elia, A. J., McInnes, R. R. and van der Kooy, D. (2000).
Retinal stem cells in the adult mammalian eye. Science
287,2032
-2036.
Wang, Y., Macke, J. P., Abella, B. S., Andreasson, K., Worley,
P., Gilbert, D. J., Copeland, N. G., Jenkins, N. A. and Nathans, J.
(1996). A large family of putative transmembrane receptors
homologous to the product of the Drosophila tissue polarity gene frizzled.
J. Biol. Chem. 271,4468
-4476.
Wetts, R. and Fraser, S. E. (1988). Multipotent precursors can give rise to all major cell types of the frog retina. Science 239,1142 -1145.[Medline]
Wetts, R., Serbedzija, G. N. and Fraser, S. E. (1989). Cell lineage analysis reveals multipotent precursors in the ciliary margin of the frog retina. Dev. Biol. 136,254 -263.[Medline]
Willbold, E., Rothermel, A., Tomlinson, S. and Layer, P. G. (2000). Müller glia cells reorganize reaggregating chicken retinal cells into correctly laminated in vitro retinae. Glia 29,45 -57.[CrossRef][Medline]
Wodarz, A. and Nusse, R. (1998). Mechanisms of Wnt signaling in development. Annu. Rev. Cell Dev. Biol. 14,59 -88.[CrossRef][Medline]