1 Masai Initiative Research Unit, RIKEN (The Institute of Physical and Chemical
Research), 2-1 Hirowasa, Wako-shi, Saitama 351-0198, Japan
2 Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute,
2-1 Hirowasa, Wako-shi, Saitama 351-0198, Japan
* Author for correspondence (e-mail: imasai{at}postman.riken.jp)
Accepted 26 January 2005
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SUMMARY |
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Key words: cAMP-dependent protein kinase, Cyclin D, Danio rerio, p27, Wnt, Zebrafish
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Introduction |
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In all cell types, progression of the cell cycle is regulated by different
combinations of cyclins and cyclin-dependent kinases (cdks)
(Murray, 2004). Cyclin D1 is
expressed abundantly in the developing retina and is important for progression
of the cell cycle in vertebrates (Sicinski
et al., 1995
; Fantl et al.,
1995
), including in zebrafish
(Yarden et al., 1995
). Three
major types of cdk inhibitor, Cip/Waf, Kip and INK4 family proteins, are
important regulators of exit from the cell cycle
(Galderisi et al., 2003
).
Among these, a Kip family protein, p27, is expressed in the developing retina
and plays a role in the exit of retinal progenitor cells from the cell cycle
by the inhibition of cyclin D1 (Dyer and
Cepko, 2001
; Geng et al.,
2001
). Several cell-extrinsic and -intrinsic factors regulating
the cell cycle have been identified
(Ohnuma and Harris, 2003
;
Levine and Green, 2004
;
Yang, 2004
). Wnt and Notch (N)
are cell-extrinsic regulators of the cell cycle that function upstream of
cyclin D and p27 (van Es et al.,
2003
; Radtke and Raj,
2003
). In the chick retina, a Wnt family protein, Wnt2b, is
expressed in mitotic progenitor cells and promotes cell proliferation
(Kubo et al., 2003
). There
were reports that cyclin D1 is a target of ß-catenin/LEF-1, components of
canonical Wnt signalling (Tetsu and
McCormick, 1999
; Shtutman et
al., 1999
). N signalling promotes the exit of retinal progenitor
cells from the cell cycle in Xenopus retinas
(Ohnuma et al., 2002
),
although N plays a role in the maintenance of neural stem cells in the brain
(Radtke and Raj, 2003
).
Although these factors have been identified, mechanisms underlying the exit of
retinal progenitor cells from the cell cycle still remain to be
elucidated.
In zebrafish, postmitotic cells are initially generated in the ventronasal
retina adjacent to the optic stalk, and neuronal production progresses to the
entire neural retina (Hu and Easter,
1999). Our previous study revealed that neuronal production is
initiated by the interaction between the optic stalk and the neural retina,
and that its progression to the entire neural retina is regulated by the relay
of short-range signalling (Masai et al.,
2000
). The progression of differentiation of retinal ganglion
cells (RGCs) and photoreceptors in the zebrafish retina requires the
signalling molecule Hedgehog (Hh) (Neumann
and Nuesslein-Volhard, 2000
;
Stenkamp et al., 2000
)
(reviewed by Russell, 2003
;
Pujic and Malicki, 2004
).
These observations raise the possibility that Hh signalling is a candidate for
the short-range signalling required for neuronal production. However, the most
recent study demonstrated that the expression of ath5, one of
earliest markers of neuronal production, is suppressed in only 20% of embryos
treated with the Hh inhibitor cyclopamine
(Stenkamp and Frey, 2003
).
Furthermore, the suppression of ath5 expression occurs only following
treatment with cyclopamine before invagination of the optic cup, and the
progression of ath5 expression is slightly delayed by the treatment
with cyclopamine from the stage when Hh is expressed in the retina. Thus,
Stenkamp and Frey predicted that both the initial induction and early
progression of ath5 expression may be regulated by long-range
Hh-dependent midline signalling, and that short-range Hh signalling within the
neural retina plays only a supplemental role in the progression of
ath5 expression (Stenkamp and
Frey, 2003
). Thus, it is still unclear whether Hh signalling
essentially regulates the progression of ath5 expression and neuronal
production.
Much of our knowledge about the mechanisms underlying the Hh signalling
pathway is based on its genetic studies in Drosophila (reviewed by
Ingham and McMahon, 2001;
Cohen, 2003
). Hh is released
from secreting cells and binds to the receptor protein Patched (Ptc) in
responding cells, thereby relieving the Ptc-mediated inhibition of Smoothened
(Smo), a seven transmembrane protein essential for the transduction of all
Hh-signalling activity. Activated Smo signals to the transcription factor
Cubitus interruptus (Ci), which acts as a bipotential transcription factor
that can repress, as well as activate, Hh target genes. In the absence of Hh
signalling, the repressor form is generated by proteolytic cleavage of
full-length protein, which is promoted by cAMP-dependent protein kinase
(PKA)-mediated phosphorylation. In the presence of Hh signalling, the cleavage
of Ci is inhibited and a full-length activator isoform predominates. It is the
balance between these activator and repressor forms of Ci that determines the
specific target genes that the cell expresses in response to a particular
level of Hh-signalling activity. In vertebrates, at least three Ci homologous
genes called Gli, Gli1, Gli2 and Gli3, mediate the transcriptional response to
Hh signals. Less is known about Gli family functions than is known about Ci
function. Gli1 does not undergo preteolytic cleavage and appears to be solely
an activator of the Hh response. Gli2 and possibly Gli3 have both activator
and repressor forms, although the in vivo cleavage of these proteins has not
been directly demonstrated. In vertebrates, the activity of the Gli
transcription factors is negatively regulated by PKA
(Hammerschmidt et al., 1996
),
which possibly acts to promote the cytoplasmic sequestration of Gli1 and
generate the repressor forms of Gli2 and Gli3.
Here we show that forskolin treatment effectively inhibits the progression of neuronal production in the zebrafish retina. In the presence of forskolin, a few postmitotic cells are generated adjacent to the optic stalk, but neuronal production is completely inhibited in the remaining region of the neural retina. Forskolin activates adenynyl cyclase, which increases the level of cAMP and then the activity of PKA. The introduction of a dominant-negative form of PKA (dnPKA) rescues the progression of neuronal production in the presence of forskolin. These data clearly show that the initial induction of neurogenesis in the ventronasal retina is PKA-independent, and that the activation of PKA strongly inhibits the progression of retinal neurogenesis. In the presence of forskolin, almost all retinal cells fail to exit from the cell cycle but instead continue to proliferate, suggesting that PKA inhibits the cell-cycle exit of retinal progenitor cells. The introduction of a cdk inhibitor, p27, inhibits cell-cycle progression even in the presence of forskolin, suggesting that PKA functions upstream of p27. Blockade of Wnt signalling also inhibits cell-cycle progression in forskolin-treated retinas. However, the activation of Wnt signalling promotes the cell-cycle progression even in the absence of PKA activity. These observations suggest that Wnt activation is sufficient to maintain proliferation regardless of the level of PKA activity, and that PKA seems to inhibit exit from the Wnt-mediated cell cycle rather than stimulate Wnt-mediated cell-cycle progression. Cell-cycle exit of retinoblasts is similarly affected by blockade of the Hh signalling pathway. Together, these data suggest that Hh signalling regulates the progression of neuronal production.
In Drosophila, Hh is activated in differentiating photoreceptors
and acts on adjacent uncommitted cells causing them to differentiate into
photoreceptors, which in turn become a new source of Hh signals
(Kumar, 2001). Zebrafish
sonic hh (shh) and tiggy-winkle hh (twhh)
are expressed in RGCs and induce RGC differentiation non-cell-autonomously
(Neumann and Nuesslein-Volhard,
2000
), raising the possibility that Hh emanating from RGCs induces
neighbouring progenitor cells to generate postmitotic cells, which in turn
become a source of Hh signals. However, pulse treatment with forskolin shows
that Hh regulates at least two distinct steps of RGC differentiation:
cell-cycle exit of retinoblasts and progression of postmitotic cells into
mature RGCs. This dual requirement of Hh signalling implies that the mechanism
underlying a neurogenic wave in the zebrafish retina is more complex than that
in the Drosophila eye.
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Materials and methods |
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The treatment with forskolin and cyclopamine
Embryos were treated with forskolin according to the procedure previously
described (Barresi et al.,
2000). As Hh-dependent midline signalling promotes the fate of the
optic stalk and represses that of the neural retina
(Macdonald et al., 1995
),
forskolin treatment from early stages causes the cyclopia phenotype
(Barresi et al., 2000
). To
prevent this defect, forskolin was applied to embryos after the regional
specification of the optic cup, by midline signalling, is completed. All
treatments with forskolin in this study were carried out from 18 hours
postfertilisation (hpf), except the pulse treatment shown in
Fig. 6. According to the
previous study (Neumann and
Nuesslein-Volhard, 2000
), cyclopamine (TRC Biomedical Research
Chemicals, New York, Ontario, Canada) was dissolved in ethanol and diluted to
a final concentration of 100 µM for use. We also used cyclopamine-KAAD
(Calbiochem) at a final concentration of 75 µM.
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BrdU incorporation
Dechorionated embryos were soaked for 30 minutes in Ringer's solution
containing 10 mM BrdU (Sigma) and 15% dimethyl sulfoxide (DMSO) at 6°C.
After BrdU treatment, the embryos were washed, incubated for 30 minutes in
Ringer's solution at 28.5°C and fixed with 4% paraformaldehyde (PFA). When
embryos were older than 2 days postfertilization (dpf), Ringer's solution with
10 mM BrdU was injected into the yolk of embryos. After at least a 2-hour
incubation at 28.5°C, the embryos were fixed with 4% PFA.
Labelling for apoptosis
Embryos were fixed with 4% PFA and sectioned at 8 µm on a microtome
cryostat, HM550M-OM (MICROM International GmbH). Apoptotic cells were detected
by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling
(TUNEL) using an in situ cell death detection kit (Roche).
DNA constructs for expressing dnPKA, p27, N-Tcf3 and
47-ß-catenin
The pCS2 vector (Rupp et al.,
1994) carrying mouse dnPKA was described by Ungar and Moon
(Ungar and Moon, 1996
). This
plasmid was modified to generate the expression construct pCS2[hsp:dnPKA], by
replacing the CMV promoter with a zebrafish heat-shock inducible promoter
(hsp) (Halloran et al., 2000
).
The coding region of Xenopus p27
(Su et al., 1995
) and
zebrafish
N-Tcf3 (Kim et al.,
2000
) was inserted into the pCS2MT expression vector, resulting in
the addition of the myc-tag to the N terminus of these proteins. GFP was fused
to the C terminus of the coding region of
47-ß-catenin
(Chenn and Walsh, 2002
). These
plasmids were modified to generate the expression constructs
pCS2[hsp:myc-p27], pCS2[hsp:myc-
N-Tcf3] and
pCS2[hsp:
47-ß-catenin-GFP], by replacing the CMV promoter with
zebrafish hsp.
Heat-shock treatment and BrdU labelling of embryos injected with the DNA construct
Embryos injected with the construct pCS2[hsp:dnPKA], pCS2[hsp:myc-p27] or
pCS2[hsp:myc-Tcf3] were incubated at 39°C from 17 to 18 hpf, and
soaked in water containing forskolin from 18 hpf. Embryos expressing dnPKA
were fixed with 4% PFA at 33 hpf and examined for in situ hybridisation with
the ath5 RNA probe. At 33 hpf, embryos expressing p27 or
Tcf3
were labelled with BrdU and then fixed with PFA at least 2 hours after the
BrdU incorporation. These embryos were labelled with the anti-myc antibody and
signals were visualised by staining with HRP-conjugated anti-mouse IgG
antibodies (Histofine kit, Nichirei), using 3,3'-diaminobenzidine (DAB)
(Sigma) as a substrate. These labelled embryos were sectioned using a cryostat
and the cryosections were treated with 2N HCl for 60 minutes to expose the
nuclei. After HCl treatment, sections were labelled with the anti-BrdU
antibody and BrdU-positive areas were visualised as fluorescence by labelling
with Alexa532-conjugated anti-mouse IgG antibody. HCl treatment seems to
inactivate the epitope structures of the anti-myc antibody, resulting in an
absence of cross-reactions between the Alexa532 anti-mouse IgG antibody and
anti-myc antibody.
Co-expression of dnPKA and 47-ß-catenin
A mixture of plasmids, pCS2[hsp:dnPKA] (30 µg/ml) and
pCS2[hsp:47-ß-catenin-GFP] (30 µg/ml), was injected into
embryos. Embryos were incubated at 39°C from 17 to 18 hpf, developed until
48 hpf and then fixed with 4% PFA.
Cloning of zebrafish homologues of the cdk inhibitor p27
We searched the zebrafish genomic database, which has been provided by the
Wellcome Trust Sanger Institute, and identified two zebrafish homologues of
p27. Their Ensembl gene identification numbers in this database are
ENSDARG00000020832 and ENSDARG00000010878, which are referred to as p27a and
p27b, respectively, hereafter. The sequences of p27a and p27b have been
deposited in GenBank with the Accession numbers AF398516 and BI887574 (EST
sequence), respectively. Their cDNA fragments were amplified by PCR using
primers specific to their 5' and 3' sequences. The phylogenetic
tree was constructed on the basis of the full-length amino acid sequences
using the GENETYX-MAC program based on the Neighbour-Joining (NJ) method
(Software Development).
Calculation of the ratio of BrdU-positive area to total area within the neural retina
Cryosections labelled with the anti-BrdU antibody were scanned under a LSM
510 laser-scanning microscope (Carl Zeiss). Using NIH Image, BrdU signals were
converted to a binary scale with two digits, 0 (negative) and 1 (positive), by
which the BrdU-positive area is adjusted to the outlines of BrdU-positive
cells. This procedure approximates the ratio of the BrdU-positive area to the
total area to the ratio of BrdU-positive cell number to total cell number. The
number of pixels corresponding to 1 and 0 within the neural retina was
determined. The ratio of the BrdU-positive area to the total area was
calculated as the ratio of the number of 1 pixels to the number of 1+0
pixels.
Morpholino oligonucleotide injection
Morpholinos were purchased from Gene Tools. Anti-sense morpholino
oligonucleotides for shh (shh-MO), twhh (twhh-MO),
Gli1 (Gli1-MO) and Gli2 (Gli2-MO) genes were designed as
previously described (Nasevicius and
Ekker, 2000; Karlstrom et al.,
2003
). A mixture of shh-MO (1 mg/ml) and twhh-MO (1 mg/ml), or a
mixture of Gli1-MO (1 mg/ml) and Gli2-MO (1 mg/ml), was injected into the
yolk. Injected embryos were incubated until they reached the appropriate stage
and then fixed with 4% PFA for in situ hybridisation, plastic section or
antibody labelling.
Assessment of severity of the progression of ath5 expression in the blockade of Hh signalling
According to the severity of the defect in ath5 expression,
embryos labelled with ath5 RNA probe were classified into four
groups: no expression, severe, mild and normal. Severe means that
ath5 expression is initiated in the ventronasal retina but fails to
spread to the dorsal and temporal retina. Mild means that the progression of
ath5 expression occurs to some degree but does not reach the whole of
the neural retina. Typical examples are shown in
Fig. 4L-L''.
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Results |
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To exclude the possibility that postmitotic cells die immediately after their generation in the presence of forskolin, their cell death pattern was examined by labelling with Acridine Orange. Apoptosis was observed in both forskolin-treated and control DMSO-treated retinas but its spatiotemporal cell death pattern did not correlate with the pattern of progression of ath5 expression (data not shown). Even though their cell death pattern does not correlate to the pattern of progression of ath5 expression, forskolin treatment has an effect on the maintenance of retinal cells. TUNEL analysis of cryosections revealed that at 33 hpf the number of apoptotic cells in the presence of forskolin was more than three times higher than that in the presence of DMSO (Fig. 2G,H,J), suggesting that forskolin treatment affects the survival of mitotic retinal cells. From 33 to 48 hpf, the number of apoptotic cells increased not only in forskolin-treated retinas but also in DMSO-treated retinas (Fig. 2J), suggesting that retinal cells are sensitive to DMSO after 33 hpf.
PKA inhibits the exit of retinoblasts from Wnt-dependent cell-cycle progression
To elucidate whether PKA inhibits cell-cycle exit upstream of cyclin D1 and
p27, we examined the expression of these genes in forskolin-treated retinas.
To identify zebrafish p27 genes, we searched the zebrafish genome database and
found that there are at least two p27 homologues in zebrafish, which are
designated p27a and p27b (see Materials and methods). The phylogenetic
relationship among the p27 family proteins revealed that p27a is similar to
mouse and human p27, whereas p27b is more similar to Xenopus p27
(Fig. 3A). In situ
hybridisation analysis revealed that p27b is expressed in the
zebrafish retina, whereas p27a is expressed in the lens rather than
in the neural retina (data not shown). p27b expression was probably
localised in mitotic retinoblasts and early differentiating neurons
(Fig. 3B; data not shown).
p27b expression was markedly weak or absent in forskolin-treated
retinas (Fig. 3C). The
expression of cyclin D1 was downregulated in differentiating neurons
and localised in the CMZ in wild-type retinas
(Fig. 3D). However, cyclin
D1 expression was not downregulated and remained in a large area of the
forskolin-treated retina (Fig.
3E). We examined the transcription level of cyclin D1 by
the quantitative PCR analysis and found that the transcription level of
cyclin D1 is relatively normal in forskolin-treated heads (data not
shown), suggesting that the aberrant increase in cyclin D1 expression
does not occur in the forskolin-treated retina. Furthermore, ectopic
introduction of Xenopus p27 inhibited cell-cycle progression in the
forskolin-treated retina (Fig.
3F; 100%, n=15 retinal columns), although almost all p27
non-expressing cells were BrdU-positive. These data suggest that PKA inhibits
the cell-cycle exit of retinal cells upstream of the interaction between
cyclin D1 and p27.
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Recently, we found that proliferation is enhanced in the zebrafish retina
by the introduction of 47-ß-catenin, which lacks N-terminal
phosphorylation sites and functions as a constitutively active form of
ß-catenin (Chenn and Walsh,
2002
) (M.Y. and I.M., unpublished)
(Fig. 3H). To elucidate whether
PKA activity is required for Wnt-mediated proliferation, we examined retinal
phenotypes of embryos injected with a mixture of
47-ß-catenin and
dnPKA. The introduction of dnPKA did not suppress the hyperproliferation
induced by
47-ß-catenin (Fig.
3I), suggesting that a high level of Wnt activity can overcome the
loss of PKA activity to promote proliferation. These data suggest that Wnt
signalling is epistatic to PKA in the proliferation of retinal cells.
To elucidate whether Wnt signalling is activated by PKA, we used TOPdGFP
transgenic fish carrying GFP under the control of a ß-catenin responsive
promoter (Dorsky et al., 2002).
In TOPdGFP fish, GFP is expressed in the CMZ at 2 dpf
(Fig. 3J), suggesting that the
activation of Wnt signalling occurs in proliferating retinal cells. Forskolin
treatment did not enhance this GFP expression in the neural retina
(Fig. 3K), suggesting that PKA
does not activate the Wnt signalling pathway. Together, these data suggest
that PKA inhibits exit from the Wnt-mediated cell cycle, rather than
stimulating cell-cycle progression through the activation of Wnt
signalling.
The cell-cycle exit of retinal mitotic cells is affected in the blockade of Smo and Gli functions
A previous study demonstrated that the induction and progression of
ath5 expression are variably perturbed in the zebrafish smo
mutant slow-muscle-omitted (smu)
(Stenkamp and Frey, 2003).
Here, we show that the cell-cycle exit of retinal progenitor cells is
perturbed in smu mutants. In
smub577/ retinas, lamination was usually
delayed (Fig. 4B,B'). In
the smub577 mutant, the initial induction of ath5
expression occurred in the presumptive ventronasal retina, but the progression
of ath5 expression was variably affected among
smub577/ embryos from severe
(n=6/21, Fig. 4D) to
mild (n=6/21, Fig.
4D'). The delay of ath5 expression became less
severe in the smub577 mutant in later stages, and
ath5 expression occurred in a large region of the neural retina until
3 dpf (data not shown). In 2-dpf wild-type retinas, the anti-BrdU antibody
strongly labelled retinal cells in the CMZ and the outer layer, but not in the
RGC and inner layers, where the majority of neuronal cells were born until 2
dpf (Fig. 4E,E'). The
ratio of BrdU-positive cells relative to the total number of cells was about
30% on average in wild-type retinas, which remained constant from the nasal to
temporal regions within the optic cup (Fig.
4G). In the severe cases of smub577 mutants, a
large number of retinal cells were BrdU-positive
(Figs. 4F,F'). On
average, 70% of cells were BrdU positive in severe cases, more than twice that
in wild-type embryos. BrdU labelling of serial sections of the
smub577/ retina revealed that more than 80%
of cells in the temporal region were mitotic, with the highest ratio of
mitotic cells being found in the nasal-temporal axis
(Fig. 4G). Since the wave of
neuronal production spreads from the nasal to temporal retina, the high ratio
in the temporal retina suggests that the progression of neuronal production is
delayed in smub577/ retinas.
The progression of retinal neurogenesis is more severely inhibited in
embryos treated with forskolin than in the smu mutant. The difference
between forskolin treatment and the genetic blockade of Smo function might be
the level of PKA activity. One of the major substrates of PKA is the
cAMP-responsible element binding factor (CREB)
(Lonze and Ginty, 2002).
Because PKA phosphorylates Ser133 of CREB, we examined the level of this
phosphorylation in forskolin-treated and smu mutant embryos. Western
blot analysis with an antibody against phosphorylated Ser133 of CREB revealed
that the level of CREB phosphorylation is much higher in forskolin-treated
embryos than in control DMSO-treated embryos
(Fig. 4H). By contrast, the
level of CREB phosphorylation was not elevated in smu mutants
(Fig. 4H). These data suggest
that high activation of PKA occurs only after forskolin treatment.
As PKA inhibits the Hh signalling pathway by generating the repressor form of Gli, the repressor activity of Gli may be higher in the presence of forskolin than when Smo function is blocked. If this is the case, the level of the Gli repressor activity may correlate with the severity of the defect in retinal neurogenesis. An alternative possibility is that PKA activates Hh-independent signalling pathways, such as CREB, which might contribute to the forskolin-induced phenotype. To distinguish between these possibilities, we examined retinal phenotypes caused by the inhibition of Gli proteins, which function downstream of PKA. To inhibit Gli function, embryos were injected with a mixture of morpholino antisense oligonucleotides of Gli1 and Gli2 (Gli-MO). In Gli-MO-injected embryos, retinal lamination was severely delayed, as in the smu mutant (Fig. 4I,J). In Gli-MO-injected retinas, the progression of ath5 expression was variably perturbed from no expression (n=12/63, Fig. 4L) to severe (n=36/63, Fig. 4L'), mild (n=5/63, Fig. 4L'') and almost normal (n=10/63, data not shown) expression. The severity of the progression defect in Gli-MO-injected embryos is intermediate between that observed in forskolin-treated embryos and that seen in smu mutant embryos (Fig. 4M). These data suggest that the blockade of Gli functions causes defects in the cell-cycle exit of retinal progenitor cells. Together, these data suggest that PKA inhibits Hh-mediated Gli activation, which regulates the progression of neuronal production.
A wave of ath5 expression spreads to the temporal retina earlier than that of shh:GFP expression
Zebrafish Shh and Twhh are expressed in RGCs and induce RGC differentiation
in neighbouring uncommitted areas (Neumann
and Nuesslein-Volhard, 2000), raising the possibility that Hh
produced by RGCs promotes mitotic retinoblasts to generate neurons. If this is
the case, the progression of ath5 expression must spatially and
temporally correlate with that of RGC differentiation. However, several
contradictory observations have been reported. Because the first RGCs start to
extend their axons at 28 hpf (Laessing and
Stuermer, 1996
; Schmitt and
Dowling, 1996
), there is at least a 3-4 hour time lag between
ath5 expression and this first morphological appearance of RGC
differentiation. Furthermore, cyclopamine treatment after 26 hpf completely
inhibits the progression of RGC differentiation
(Neumann and Neusslein-Volhard,
2000
), although the progression of ath5 expression is
only mildly delayed by treatment with cyclopamine after 27 hpf
(Stenkamp and Frey, 2003
).
These observations suggest that the progression of ath5 expression
does not strongly correlate with that of RGC differentiation. To elucidate why
this lack of correlation occurs, we examined ath5 and shh
expression in more detail. shh expression was monitored by GFP
expression under the control of shh retinal enhancers (referred to as
shh:GFP). shh:GFP was expressed in the ventral forebrain only, and was not in
the eye until at least 27 hpf (data not shown). shh:GFP expression was
detected in a few cells in the ventronasal retina adjacent to the optic stalk
at 29 hpf (Fig. 5A). Faint
expression of shh:GFP progressed to the dorso-nasal retina at 31 hpf
(Fig. 5B). shh:GFP expression
spread to the temporal retina at 35 hpf
(Fig. 5C). This expression
profile is consistent with previous published data
(Neumann and Nuesslein-Volhard,
2000
), and suggests that shh:GFP expression correlates with the
maturation of RGCs.
To facilitate the detection of ath5 expression with high
sensitivity, we used the transgenic line Tg(ath5:GFP)
(Masai et al., 2003). BrdU
labelling of retinas expressing ath5:GFP showed that ath5:GFP-expressing cells
were BrdU-negative, suggesting that ath5:GFP is a marker of newly-generated
postmitotic cells (Fig. 5D).
Similar to the results of in situ hybridisation, ath5:GFP expression started
in the ventronasal retina at 25 hpf (Fig.
5E). Compared with in situ hybridisation, it was more clearly
demonstrated that the wave front of ath5 expression had already
reached the temporal retina at 27 hpf, although the density of GFP-positive
cells was low (Fig. 5F). At 31
hpf, the area where ath5:GFP-positive cells were located became slightly
larger and the number of ath5:GFP-positive cells increased
(Fig. 5G). These data suggest
that the wave of ath5:GFP expression progresses earlier than that of shh:GFP
expression. To exclude the possibility that the time lag between ath5:GFP and
shh:GFP is due to the difference of transgenic strains, we confirmed that
ath5 mRNA expression spreads to the temporal retina at 29 hpf in
shh:GFP transgenic embryos (Fig.
5H).
Forskolin treatment inhibits two distinct steps for RGC differentiation
The profile of ath5:GFP expression suggests that Hh signalling
present before 27 hpf regulates the progression of neuronal production. To
determine the critical period when Hh signalling is required for the
progression of ath5 expression, forskolin was applied within
different time windows (Fig.
6M). To estimate the time lag from the forskolin treatment to the
inhibition of Hh signalling pathway, we monitored ptc1 expression
during and after the forskolin pulse treatment. The expression of
ptc1 is regulated by Hh signalling and occurs in the ventral part of
CNS at 24 hpf (Fig. 6A)
(Concordet et al., 1996). Two
hours after the start of forskolin treatment, ptc1 expression
severely decreased (n=5/5, Fig.
6B), suggesting that Hh signalling is suppressed within 2 hours of
forskolin treatment (Fig. 6E).
Two hours after the removal of forskolin, ptc1 expression remained
downregulated (n=6/6, Fig.
6C). By 4 hours after the removal of forskolin, ptc1
expression had increased but was not fully recovered (data not shown). By 6
hours after the removal of forskolin, ptc1 expression levels had
recovered to the levels seen in control DMSO-treated embryos (n=6/6,
Fig. 6D). These data suggest
that Hh signalling is recovered between 2 and 6 hours after the removal of
forskolin (Fig. 6E).
Here we monitored ath5:GFP and shh:GFP as markers of newly generated neurons and mature RGCs, respectively. Forskolin treatment before 22 hpf had no effect on the progression of ath5:GFP expression (Fig. 6F). Treatment from 18 to 24 hpf induced various phenotypes of ath5:GFP expression (Fig. 6G-G''), suggesting that forskolin treatment around 24 hpf inhibits the progression of ath5:GFP expression. Indeed, forskolin treatment between 22 and 26 hpf completely inhibited the progression of ath5:GFP expression (Fig. 6H). Interestingly, forskolin treatment after 27 hpf failed to block the progression of ath5:GFP expression (Fig. 6J) but completely inhibited the progression of the expression of shh:GFP (Fig. 6K) and that of another RGC marker, islet1 (data not shown). To exclude the possibility that it is the difference in the transgenic strains that causes a different response to forskolin treatment between ath5:GFP and shh:GFP expression, we confirmed that ath5 mRNA spreads to the large region of the neural retina in the Tg(shh:GFP) embryo shown in Fig. 6K (Fig. 6L). Together, these data suggest that Hh signalling regulates two distinct steps of RGC differentiation: cell-cycle exit of retinoblasts and the progression of ath5-positive immature neurons to mature RGCs. Considering the time lag between forskolin treatment and inhibition of the Hh signalling pathway (Fig. 6N), Hh signalling must regulate the progression of ath5 expression between 24 and 29 hpf. Hh signalling after 29 hpf is not essential for the progression of ath5 expression but is still required for the progression of RGC maturation.
Morpholino-antisense oligonucleotides for shh and twhh inhibits the wave of neuronal production
Pulse treatment with forskolin suggests that the wave of ath5
expression is regulated by Hh activity that is present before 29 hpf. shh:GFP
expression is initiated at 29 hpf (Fig.
5A). It was reported that shh RNA starts to be expressed
at 28 hpf (Neumann and Nuesslein-Volhard,
2000). These observations raise the possibility that there is a
very weak expression of shh and twhh in 24- to 29-hpf
retinas, which is sufficient to regulate the wave of ath5 expression.
Alternatively, it is possible that Hh proteins other than Shh and Twhh
regulate the progression of ath5 expression. A third possibility is
that ath5 expression is not regulated by the short-range action of Hh
signalling within the neural retina, but by the long-range action of Hh
expressed in other tissues.
To investigate whether inhibition of the Shh- and Twhh-mediated pathway has an effect on the progression of ath5 expression, we examined the phenotypes of embryos injected with a mixture of morpholino-antisense oligonucleotides for Shh and Twhh (hh-MO). In hh-MO-injected retinas, the progression of ath5 and islet1 expression was perturbed (Fig. 7B,D). Retinal cells in hh-MO-injected embryos largely incorporated BrdU (Fig. 7F). The severity of the defect in ath5 expression in hh-MO-injected embryos was similar to that in Gli-MO-injected embryos (Fig. 7G, compare with Fig. 4M), suggesting that Shh and Twhh regulates the progression of ath5 expression. To elucidate whether the local depletion of Shh and Twhh activities within the neural retina affects ath5 expression, hh-MO-injected donor cells were transplanted into wild-type host retinas. In this experiment, to facilitate the detection of ath5 expression, we used the transgenic line Tg(ath5:GFP). ath5:GFP expression was rarely observed in retinal cells derived from hh-MO-injected donor cells (Fig. 7H,I; n=5/5 embryos), and in some cases, ath5:GFP was not detected in wild-type cells located adjacent to the temporal side of hh-MO-injected cells (Fig. 7J), suggesting that Hh activity in the neural retina is necessary to regulate the progression of ath5 expression. Taken together, these data suggest that short-range Shh and Twhh signalling regulates the wave of neuronal production in the zebrafish retina.
|
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Discussion |
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PKA inhibits cell-cycle exit of retinoblasts through interaction with both Hh and Wnt signalling
In the presence of forskolin, cyclin D1 expression is not
downregulated and p27 expression severely decreases. Furthermore, the
introduction of p27 inhibits cell-cycle progression even in the presence of
forskolin. These data suggest that PKA promotes cell proliferation upstream of
the interaction between cyclin D1 and p27. It was reported that cyclin D1 is a
direct target of Wnt/ß-catenin signalling
(Tetsu and McCormick, 1999;
Shtutman et al., 1999
). We
found that
47-ß-catenin promotes the proliferation of retinal
cells in zebrafish, and that
N-Tcf3 inhibits the proliferation of
retinal cells induced by forskolin treatment. These data suggest that
Wnt/ß-catenin signalling is required for PKA-mediated proliferation.
However, dnPKA does not suppress Wnt-induced overproliferation, suggesting
that a high level of Wnt activity maintains cell proliferation in the absence
of PKA activity. Together, these data suggest that PKA plays a supplemental
role in Wnt-induced proliferation, or that PKA inhibits exit from Wnt-mediated
cell-cycle progression rather than promoting cell-cycle progression in concert
with Wnt signalling.
What is the molecular mechanism that underlies PKA-mediated inhibition of
retinal neurogenesis? There are two possible pathways
(Fig. 8A). One possibility is
that PKA inhibits the expression of the cdk inhibitor p27. Because
PKA antagonises the Hh signalling pathway, Hh-mediated Gli activation may be
required for p27 expression in the zebrafish retina. Although it is
unknown whether Gli transcription factors directly regulate the transcription
of p27, the activator form of Gli may positively regulate the
expressison of p27. It was reported that Indian Hh induces colonic
epithelial differentiation by antagonising Wnt/ß-catenin signalling
(van den Brink et al., 2004).
In this case, Hh signalling is required for activation of the cdk inhibitor
Cip1/Waf1/p21, and negatively regulates cyclin D1, suggesting that a common
mechanism may regulate both zebrafish retinal neurogenesis and mouse colonic
epithelial differentiation. Although Gli-MO injection induces severe defects
in the progression of neuronal production, it is also possible that PKA
promotes cell proliferation though Gli-independent pathway. One of the major
PKA substrates, CREB, binds to the promoter of cell-cycle regulators such as
cyclin D1 (Lonze and Ginty,
2002
). PKA phosphorylates Ser133 of CREB and this phosphorylated
form of CREB recruits p300/CBP histone acetyltransferase (HAT) to activate the
transcription of cyclin D1. As
N-Tcf3 inhibits cell-cycle
progression in the presence of forskolin, phophorylated CREB may activate the
transcriptional activity of Tcf3/ß-catenin complex in this model. We show
that forskolin treatment does not activate transcription under the control of
the ß-catenin responsive promoter
(Fig. 3K). However, this is not
contradictory to this model, because the ß-catenin responsive promoter
does not contain the CREB-binding sequence. In the developing mouse
cerebellum, Shh induces G1-S transition to promote proliferation of granule
cell precursors (Wechsler-Reya and Scott,
1999
), whereas an extracellular matrix protein, Vitronectin,
promotes granule cell differentiation through PKA-mediated CREB
phosphorylation (Pons et al.,
2001
). As Shh suppresses PKA-mediated CREB phosphorylation in
cerebellar development, competition between the Shh and PKA-CREB pathway
determines whether granule cell precursors continue to proliferate or whether
they differentiate. Although the role of Hh in cerebellar development is the
reverse of that in zebrafish retinal development, the PKA-CREB pathway may be
involved in the switch between proliferation and differentiation in the
zebrafish retina.
|
Hh signalling regulates two distinct steps of RGC differentiation
A previous study suggested that shh is expressed in RGCs and
induces neighbouring uncommitted cells to differentiate into RGCs
(Neumann and Nuesslein-Volhard,
2000). In this study, we show that Hh signalling promotes mitotic
retinoblasts to generate postmitotic daughter cells. These results raise the
possibility that Hh signals emanating from RGCs induce adjacent mitotic cells
to generate neurons that become RGCs and function as a new source of Hh
signals. However, the wave front of ath5 expression reaches the
temporal retina at 27 hpf, before the first differentiated RGC starts
axonogenesis (Laessing and Stuermer,
1996
), which suggests that mature RGCs are not the source of Hh
signals. Furthermore, this early spread of ath5 expression seems
contradictory to the previous observation that blockade of Hh signalling by
cyclopamine after 26 hpf strongly inhibits the progression of RGC
differentiation (Neumann and
Nuesslein-Volhard, 2000
). These data suggest that the wave of
ath5 expression does not strongly correlate with that of RGC
differentiation. In this study, we show that forskolin treatment after 27 hpf
has no effect on the progression of ath5 expression, but that it does
inhibit the progression of markers of mature RGCs. These data suggest that Hh
signalling regulates two distinct steps of RGC differentiation: the cell-cycle
exit of retinoblasts and the progression of ath5-positive neurons
into mature RGCs. Considering the time lag between forskolin treatment and the
inhibition of Hh signalling, Hh signalling after 29 hpf does not regulate the
progression of ath5 expression but does regulate the progression of
RGC differentiation (Fig. 6N).
After 29 hpf, Hh may promote the progression of uncommitted postmitotic
neurons into mature RGCs. This later requirement of the Hh signalling pathway
for RGC differentiation is consistent with the previous observation of Neumann
and Nuesslein-Volhard (Neumann and
Nuesslein-Volhard, 2000
).
In this study, we show that only forskolin treatment completely inhibits
the progression of neuronal production; the progression of neuronal production
is only delayed following cyclopamine treatment or in the smu
mutation (Stenkamp and Frey,
2003). However, it was reported that the progression of RGC
maturation is severely inhibited by treatment with cyclopamine
(Neumann and Nuesslein-Volhard,
2000
). These observations suggest that mild blockade of Hh
signalling is sufficient to inhibit the progression of RGC maturation, whereas
severe blockade of Hh signalling is necessary to inhibit the progression of
neuronal production. If this is the case, it is possible that a low dose of Hh
signals is sufficient to induce the cell-cycle exit of retinoblasts, while a
high dose of Hh signals is necessary to promote the progression of
ath5-positive cells into mature RGCs. This idea seems to be
consistent with a model proposing that early differentiating cells near the
wave front of neuronal production express a low level of Hh, which acts on
adjacent retinoblasts causing them to exit from the cell cycle, and that
mature RGCs express a high level of Hh, which acts on adjacent uncommitted
neurons causing them to differentiate into mature RGCs
(Fig. 8B).
What is the significance of such dual regulation of Hh signalling in RGC
differentiation? Initial Hh signalling generates ath5-positive
neurons, which might be uncommitted to any retinal cell-types. Later Hh
signalling promotes ath5-positive neurons to differentiate into RGCs.
It is possible that some ath5-positive neurons do not respond to the
later Hh signals and differentiate into other types of retinal cells, such as
amacrine cells. If this is the case, the dual actions of Hh play a role in the
generation of diversity of retinal cell-types through the maintenance of a
pool of uncommitted neurons. As another possibility, a pool of uncommitted
neurons may be useful to regulate the number of RGCs during development. The
most recent study revealed that Shh is expressed in amacrine cells and directs
the differentiation and lamination of the inner and outer nuclear layers
(Shkumatava et al., 2004).
Although the authors showed that Shh expression in amacrine cells is
independent of RGC differentiation, it is unknown whether Shh expression in
amacrine cells requires the initial action of Hh signals that mediate
cell-cycle exit of retinoblasts. In the future, it will be important to
elucidate whether shh-expressing amacrine cells are generated from a
pool of ath5-positive neurons.
Short-range action of Shh and Twhh regulates the progression of ath5 expression in zebrafish
Pulse treatment results suggest that Hh signalling between 24 and 29 hpf is
required for the wave of ath5 expression
(Fig. 6N). Furthermore, the
introduction of shh and twhh morpholino-antisense
oligonucleotides blocks neuronal production in the retina. These data suggest
that Shh and Twhh regulate the progression of ath5 expression between
24 and 29 hpf. However, it is difficult to detect shh and
twhh mRNA expression in the neural retina at this early stage,
although it was reported that shh RNA is expressed in the retina at
28 hpf (Neumann and Nuesslein-Volhard,
2000). One possibility is that Shh and Twhh expressed in the
ventral forebrain may have a long-range action on progenitor cells of the
optic cup. It was reported that Hh expressed in midline tissue is important
for proliferation of the developing forebrain in chicks and mice, probably
through its long-range actions (Britto et
al., 2002
; Ishibashi and
McMahon, 2002
). The most recent study on Hh signalling in the
zebrafish retina also suggested that Hh signalling outside the optic cup
regulates ath5 expression before 27 hpf
(Stenkamp and Frey, 2003
).
However, we do not consider this as the most likely explanation, as the wave
of ath5 expression normally occurs when the optic cups are dissected
from the forebrain at 18 hpf, and cultured as an explant later
(Masai et al., 2000
),
suggesting that a source of Hh signals is localised within the optic cup.
Furthermore, when the dissected eye cup was divided into two (the nasal and
temporal halves) only the nasal half expressed ath5
(Masai et al., 2000
),
suggesting that short-range Hh signalling acts from the nasal to temporal
regions across the neural retina. Transplantation of hh-MO-injected cells into
wild-type host retinas demonstrated that ath5 expression is rarely
observed in hh-MO-injected retinal columns, and that wild-type cells fail to
express ath5 when they are located adjacent to the temporal side of
Shh- and Twhh-deprived cells. These data suggest that a short-range action of
Shh and Twhh expressed in the neural retina regulates the wave of
ath5 expression and neuronal production. Low levels of Shh and Twhh
expression may spread to the temporal retina up until 27 hpf and may be
sufficient to regulate the wave of ath5 expression.
Does Hh function as a mitogen or an anti-mitogen in the vertebrate retina?
In this study, we propose that Hh signals induce the cell-cycle exit of
retinal progenitor cells. However, several studies have suggested that Hh
functions as a mitogen for neural stem cells in the retina and in the brain,
for astrocyte precursor cells in the optic stalk, and for granule cell
precursors in the cerebellum (Roy and
Ingham, 2002; Ruiz i Altaba et
al., 2002
). The observation of such an opposite phenotype may be
due to the difference in cell types or the species used in the studies.
Otherwise, as discussed previously (Yang,
2004
), the difference in the dose of Hh signals may cause the
opposite behaviour of retinal progenitor cells. For example, a low dose of Hh
signals as an anti-mitogen promotes neuronal production, whereas a high dose
of Hh signals inhibits the differentiation of early-born cell types to
maintain a pool of mitotic cells for the generation of late-born cell types.
In Drosophila eyes, Hh regulates not only neuronal differentiation by
inducing the proneural gene atonal, but also proliferation by
inducing cyclin D/E
(Duman-Scheel et al., 2002
).
Such dual roles of Hh as a mitogen and an anti-mitogen may coordinate
proliferation with cell differentiation in the vertebrate retina.
The mitogenic role of Hh has been proposed from in vitro experiments
carried out using cell pellets or explant culture. Recent in vivo analyses of
the role of Hh in the vertebrate retina showed different phenotypes of retinal
neurogenesis. In conditional shh knock-out mice, Müller glial
cells fail to differentiate properly and the outer photoreceptor layer shows a
rosette structure (Wang et al.,
2002). In frog retina treated with the Hh inhibitor cyclopamine,
retinal progenitor cells are normal and only differentiation of the pigmented
epithelium is perturbed (Perron et al.,
2003
). We could not detect any defects in the differentiation of
the pigment epithelium in zebrafish embryos treated with forskolin or in
smu/ embryos. The roles of Hh signalling in
retinal development may be diverse among different species of vertebrates. In
the future, it will be important to elucidate why retinal cells show such
different behaviours in response to Hh signals.
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ACKNOWLEDGMENTS |
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