1 Department of Anatomy, University of Cambridge, Downing Street, Cambridge,
UK
2 Developmental Biology, Dipartimento di Fisiologia e Biochimica, Laboratorio di
Biologia Cellulare e dello Sviluppo, Universita di Pisa, Ghezzano, Pisa,
Italy
* Author for correspondence (e-mail: harris{at}mole.bio.cam.ac.uk)
Accepted 10 December 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Retinal specification, Cell fate, otd, Otx, XOtx2, XOtx5, XOtx5b, Bipolar cell, Photoreceptor, VP16, Engrailed, Xenopus laevis, Opsin, Lipofection
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The link between photoreceptors and bipolars is further demonstrated by
fate switching experiments. A late precursor may differentiate either as a
photoreceptor or a bipolar depending on environmental factors. For example,
exposing rat retinal cell cultures to the cytokine, ciliary neurotrophic
factor (CNTF), causes a dramatic decrease in the number of opsin-positive
cells and a compensatory increase in the number of cells expressing bipolar
cell-specific markers, suggesting a change in cell fate choice from rods to
bipolars (Ezzeddine et al.,
1997). Similarly, when rat retinal cells are cultured at low
density there is a decrease in the number of opsin-positive cells and an
increase in the number of cells expressing the bipolar cell marker, 115A10
(Altshuler and Cepko, 1992
).
Co-culturing late with early progenitors leads to a similar shift in cell
fate, which can be blocked by the CNTF antagonist, hLIF05
(Belliveau et al., 2000
). The
sensitivity of late retinal precursors to extrinsic factors such as CNTF
demonstrates that external cues influence the internal switches that
differentially specify the fate of these two cell types.
We have found that Xenopus photoreceptors and bipolars also
express, XOtx5b, which is a member of the otd/Otx family of
paired-like homeodomain transcription factors
(Vignali et al., 2000).
otd (also known as oc; ocelliless) is the only
otd/Otx family member identified in Drosophila and it
contributes to head formation (Finkelstein
et al., 1990
). The vertebrate homologues of otd include
Otx1, Otx2, Otx3, Otx4, Otx5, Otx5b (96% similar to Otx5) and
Crx, which are all involved in anterior embryo and sensory organ
formation (Acampora et al.,
1998a
; Acampora et al.,
1998b
; Acampora et al.,
1996
; Acampora and Simeone,
1999
; Andreazzoli et al.,
1997
; Bovolenta et al.,
1997
; Furukawa et al.,
1997
; Gammill and Sive,
1997
; Kablar et al.,
1996
; Kuroda et al.,
2000
; Martinez-Morales et al.,
2001
; Pannese et al.,
1995
; Sauka-Spengler et al.,
2001
; Suda et al.,
1999
; Vignali et al.,
2000
). The first vertebrate homologues of otd, Otx1 and
Otx2, were characterised in mouse
(Simeone et al., 1992
).
Otx2-/- mice lack fore- and midbrain structures, while a
null mutation in Otx1 is less severe, causing, among other defects, a
reduction in brain size and lack of ciliary process next to an otherwise
normal retina (Acampora et al.,
1998a
; Acampora et al.,
1996
; Acampora and Simeone,
1999
; Martinez-Morales et al.,
2001
). Overexpression of Xenopus Otx2 (XOtx2)
suggests that it plays a similar role in frog
(Andreazzoli et al., 1997
;
Blitz and Cho, 1995
;
Kablar et al., 1996
;
Pannese et al., 1995
).
XOtx5b is also expressed during early embryogenesis. Overexpression
of XOtx5b, like XOtx2, produces ectopic cement gland and
neural tissue as well as gross embryonic abnormalities in which posterior
structures are absent or reduced and the neural tube fails to close
(Kablar et al., 1996
;
Pannese et al., 1995
;
Vignali et al., 2000
). Because
of their roles in early embryonic development, the functions of Xenopus
XOtx5b and XOtx2 in retinal differentiation have not been
previously determined.
Crx was isolated in a yeast one-hybrid screen using the rhodopsin
promoter as bait (Chen et al.,
1997; Furukawa et al.,
1997
). Crx can bind the rhodopsin promoter and transactivate its
expression, along with a number of other photoreceptor-specific genes
(Chen et al., 1997
;
Livesey et al., 2000
). It is
expressed in pinealocytes as well as rod and cone photoreceptor cells. The
orthological relationships of the mammalian Crx genes with other
gnathostome otd-related genes remain controversial. Phylogenetic
analyses of the otd/Otx family members, however, support a
relationship between the Otx5/5b genes characterised in amphibians or
chondrichthyans, and the Crx genes isolated in mammals and in
zebrafish (Germot et al.,
2001
; Sauka-Spengler et al.,
2001
). These results have led to the hypothesis that
Otx5/5b and Crx genes might belong to a single orthology
class. The identification in the zebrafish of a second, unambiguously
Otx5-related, gene has challenged this view, suggesting that two
distinct orthology classes may be present in gnathostomes
(Gamse et al., 2001
). However,
novel phylogenetic analyses including a much wider range of amniote
Otx5 or Crx-related sequences as well as pufferfish
sequences, indicate that the zebrafish Otx5 and Crx genes
have arisen through a gene duplication, which occurred in the actinopterygian
lineage, and support the hypothesis of a single gnathostome Otx5/Crx
class, with an acceleration of evolutionary rate of what became Crx
early in mammalian evolution (S. Mazan, personal communication). This raises
the question of whether XOtx5b in lower vertebrates performs the same
function as Crx in mammals.
Crx, Otx1, Otx2 and Otx5b are all expressed in the
developing retina prior to retinal differentiation. Some otd/Otx
family members are expressed in both photoreceptors and bipolar cells while
others are expressed in one cell type or the other. Zebrafish Crx
(Liu et al., 2001), mouse
Otx2 (Baas et al.,
2000
; Bovolenta et al.,
1997
) and Xenopus Otx5b (see below), for example, are
expressed by both bipolars and photoreceptors, while mouse Crx is
expressed only in photoreceptors cells
(Chen et al., 1997
;
Furukawa et al., 1997
) and
Xenopus Otx2 is found in bipolar cells but not photoreceptors
(Kablar et al., 1996
;
Perron et al., 1998
). We were
therefore interested to know what roles XOtx5b and XOtx2
play in retinal development, and specifically whether they contribute to the
switch between these two cell fates. In this study, we examined the expression
of XOtx5b and XOtx2 during development and determined the
effect of each on retinal cell differentiation. We found that both
XOtx5b and XOtx2 are involved in retinal cell fate
specification. Next, we identified the domains of these proteins that were
important in determining photoreceptor verses bipolar cell fates. Finally, we
used the Xenopus opsin promoter to examine the relationship between
XOtx5b and XOtx2 in regulating photoreceptor cell
specificity.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo lipofection
DNA isolated by Qiagen maxi preps was diluted in nuclease-free water to a
concentration of 1.5 µg/µl. These stocks were spun down for at least 10
minutes at 4°C prior to use. 1 µl of each construct was mixed with 1
µl of pCS2+ green fluorescent protein (GFP) DNA, to label
transfected cells. GFP with pCS2+ vector alone was the control. 9
µl of DoTAP (Roche) was added to 3 µg of DNA and injected into stage
17-18 embryos. At stage 41, embryos were fixed for 1 hour at room temperature,
sunk in 2% sucrose overnight at 4°C and cryostat sectioned (10 µm). The
samples were then rehydrated with two washes of 1x PBS for 5 minutes,
mounted in FluorSave (CalBioChem) containing 2% DABCO (Sigma) and dried
overnight at room temperature.
Immunocytochemistry
Cryostat sections (10 µm) were washed three times for 5 minutes in
1x PBS + 0.1% Triton X-100 (PBST), blocked 30 minutes with PBST + 5%
heat-inactivated goat serum then incubated overnight at room temperature with
primary antibody: XAP-1 (DSHB) at 1:1, bovine anti-rhodopsin at 1:500 (R2-12N;
from Paul Hargrave) and anti-calbindin (Oncogene) 1:500. Sections were washed
in PBST three times for 5 minutes then three times for 20 minutes and
incubated with a 1:500 dilution of the appropriate Cy3-conjugated secondary
antibody for 2 hours. The sections were washed again in PBST and mounted.
DNA construct generation
The construction of pCS2.XOtx5b has been described previously
(Vignali et al., 2000).
pCS2.XOtx2 was generated by PCR cloning of the XOtx2 coding
region into the EcoRI site of pCS2 with 5'XOtx2 and
3'XOtx2 (Table 1).
XOtx5bN+HD-EngR was obtained by cloning the
EcoRI/TaqI fragment of XOtx5b (amino acids (aa)
1-107) into the EcoRI site of pCS2EngN. XOtx2N+HD-VP16 was
obtained by PCR cloning the XOtx2 region spanning aa residues 1-109 into the
ClaI site of pCS2VP16-N with 5'ClaX2 and 3'ClaX2.
XOtx5N+HD-VP16 was obtained by PCR cloning the XOtx5b region spanning
aa residues 1-107 into the ClaI site of pCS2VP16-N using 5'Cla
and 3'Cla.
|
Swapped domain constructs (XOtx2/5b and
XOtx5b/2+3'UTR) were generated as follows. The
BglII/SpeI fragment of T7TSXotx2
(Pannese et al., 1995)
spanning the coding region plus 3' untranslated region (3'UTR) was
cloned into the BamHI/XbaI site of pCS2+. By
site-directed mutagenesis, an Asp718 restriction site was generated
into this clone and XOtx5b at the equivalent aa residue 61 codon,
causing the CGT sequence to change to CGG. This site allowed precise swapping
of the coding region C-terminal to aa 61. Because the homeodomains (HD) are
identical in the region between aa 61 and the polyQ stretch at the HD end,
swapping is only effective beyond aa 99 of both sequences (aa 99 to end). The
3'UTR of XOtx5b/2+3'UTR was removed by PCR cloning the coding
region of XOtx5b/2+3'UTR into the ClaI/EcoRI site of
pCS2+ using 5'Cla and 3'XOtx2, to generate
XOtx5b/2.
In situ hybridisation
Whole-mount in situ hybridisations were performed on bleached embryos
(Broadbent and Read, 1999).
Dioxigenin-labelled antisense RNA probes were generated from the full-length
XOtx2 coding sequence and the 3' untranslated region of
XOtx5b [clone 13F8 (Gawantka et
al., 1998
)] as described previously
(Shimamura et al., 1994
). In
situ hybridisation on sections was done using the same protocol with the
following modifications: rehydrated sections were fixed to slides using 100%
methanol for 10 minutes, then rinsed in 1x PBS for 2 minutes and washed
3 times for 5 minutes in PBST. Sections were treated with 20 µg/ml
proteinase K for 30 seconds with subsequent wash times reduced by half.
When in situ hybridisation was followed by immunohistochemistry, we
performed the in situ hybridisation protocol described previously
(Myat et al., 1996). After the
coloration with NCT and BCIP, we washed with PBST for 5 minutes and then
followed the immunohistochemistry protocol as described above.
BrdU experiments
To determine if XOtx5b or XOtx2 transcripts were
expressed in dividing cells, stage 41 embryos were injected with BrdU
(5-bromo-2'-deoxyuridine; Labelling and Detection Kit I, Roche) in the
gut, fixed 30 minutes later and cryostat sectioned. In situ hybridisation was
performed on sections as follows: DIG-labelled probes (2 ng/ml in
hybridisation buffer) (Shimamura et al.,
1994), were heated to 70°C for 10 minutes then incubated on
sections overnight at 60°C. The rest of the protocol was as previously
described (Myat et al., 1996
).
Following NBT/BCIP staining, sections were stained for BrdU using the protocol
below.
To determine if the XOtx5b or XOtx2 constructs effect proliferation, lipofected embryos were injected with BrdU in the gut at stage 31. Embryos were fixed and cryostat sectioned (10 µm) at stage 41. The sections were stained with both BrdU and GFP antibodies. To do this, the cryostat sections were washed with 2 N HCl for 45 minutes then neutralised with several PBST washes. The antibodies for anti-mouse BrdU and anti-rabbit GFP (Molecular Probes, Eugene, OR) were added at dilutions of 1:10 and 1:500, respectively, and incubated at 37°C for 30 minutes. After washing with three changes of PBST, secondary antibodies [Alexa 488-goat anti-rabbit (Molecular Probes) and Cy3-goat anti-mouse (Chemicon)] were added together, both at a dilution of 1:500, and incubated for 30 minutes at 37°C. The sample were again washed three times with PBST and then stained with 15 µg/µl Hoechst solution for 3 minutes at room temperature to visualise nuclei. After a final three washes in PBST the sections were mounted in FluorSave (Calbiochem) containing 2% DABCO (Sigma Genosys).
TUNEL staining
Embryos were lipofected with GFP and pCS2, XOtx2 or
XOtx5b-VP16, grown to stage 31, 33/34 or 37, fixed and cryostat
sectioned. We used Intergen's ApopTag Kit to stain apoptotic cell nuclei and
counterstained with Hoechst (15 mg/ml). Retinas with at least one lipofected,
apoptotic cell were counted. Five retinas were counted for each construct at
each stage. The percentage of apoptotic lipofected cells versus all lipofected
cells was determined.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In order to compare their relative expression patterns at a more cellular level in the retina, we performed in situ hybridisation on retinal sections. At stage 25, XOtx2 is found throughout the presumptive retinal pigment epithelium (RPE) and retina, while only a few cells in the central retina express XOtx5b (Fig. 2A,F). A few hours later, at stage 28, XOtx2 expression has narrowed to the central retina and RPE, while XOtx5b expression has expanded (Fig. 2B,G). By stage 33, XOtx2 and XOtx5b expression patterns are indistinguishable; transcripts are found throughout the developing retina except in the most peripheral regions, corresponding to the ciliary marginal zone (CMZ; Fig. 2C,H). At stage 37, XOtx2 expression is detected in all layers of the peripheral retina, but is becoming restricted in the central retina to the outer edge of the inner nuclear layer or INL (Fig. 2D). Similarly, XOtx5b expression, in the central retina is narrowed to just the outer (ONL) and inner nuclear layers (Fig. 2I). In the mature, stage 41 retina, XOtx2 is only found in the outer edge of the INL and inner edge of the CMZ (Fig. 2E). XOtx5b is expressed in the inner edge of the CMZ, the outer edge of the INL and the ONL (Fig. 2J).
|
It is difficult to identify by in situ hybridisation alone which INL
retinal cell types express XOtx2 and/or XOtx5b. In mouse,
bipolar-specific markers helped show that Otx2 is expressed in
bipolar cells (Baas et al.,
2000). Unfortunately, no bipolar cell-specific antibody is
available for Xenopus and our attempts at staining Xenopus
retinas with mouse protein kinase C
(Greferath et al., 1990
) and
Chx10 (Burmeister et al.,
1996
) antibodies were unsuccessful. The most salient
distinguishing feature of a bipolar cell, however, is its characteristic
bipolar morphology with terminal arbours in the two plexiform layers.
Therefore, we lipofected cDNA for GFP into Xenopus retinas, followed
by immunohistochemistry to GFP, to reveal the morphology of a random selection
of cells. We then performed in situ hybridisations on these lipofected retinas
using XOtx2 or XOtx5b probes to identify the cell types
expressing these otd/Otx family members
(Fig. 3). We found that
messenger RNA from both of these genes is expressed in bipolar cells
(Fig. 3A-F). Of the
GFP-labelled bipolar cells 55%±8% were co-labelled with
XOtx5b, while 82%±5% were XOtx2 positive
(Table 2). Double in situ
hybridisation with XOtx5b and XOtx2 revealed that all the
INL cells that express XOtx5b also express XOtx2 (3G-I),
indicating that 55%±8% of bipolar cells co-express XOtx5b and
XOtx2 (Table 2).
|
|
We also wanted to know which photoreceptors express XOtx5b. Using cone- or rod-specific antibodies on XOtx5b-stained in situ hybridisation sections, we found XOtx5b was expressed in all cone and rod photoreceptors (Fig. 3J-O). Thus, XOtx5b is preferentially expressed in photoreceptors and a subset of bipolar cells, while XOtx2 is preferentially expressed in bipolar cells.
XOtx5b enhances photoreceptor cell fate while XOtx2
enhances bipolar cell fate
The expression of XOtx5b in all photoreceptors and the combined
expression of XOtx5b and XOtx2 in a subset of bipolar cells
led us to hypothesise that these genes may play a role in retinal cell fate.
To test this, we lipofected stage 18 retinoblasts in vivo with either
XOtx5b or XOtx2 DNA expression constructs
(Fig. 4). GFP DNA was
co-injected with the experimental DNA in order to identify the lipofected
cells. GFP DNA co-injected with vector only DNA was used as a control.
XOtx5b significantly increased (P<0.0001) the proportion
of photoreceptors in lipofected cells (Fig.
4A compared with Fig.
4B; quantified in Fig.
4D) when compared to the control. A slight decrease in the
proportion of ganglion (P=0.0002), amacrine (P=0.047),
Müller (P=0.0016) and horizontal (P=0.014) cells was
also observed in the XOtx5b lipofected cell population, while bipolar
cell numbers were unaffected. In contrast, XOtx2 overexpression
increased the proportion of bipolar cells (P<0.0001) at the
expense of photoreceptor (P=0.003) and Müller cells
(P=0.0004; Fig. 4A compared with
4C; quantified in Fig.
4E). Staining of XOtx5b lipofected retinal sections with
the XAP-1 (anti-photoreceptor) antibody confirmed that the cells counted were
indeed photoreceptors (data not shown).
|
XOtx5b lipofected photoreceptors are both rods and
cones
Since XOtx5b is normally expressed in both rod and cone
photoreceptors (Fig. 3J-O), we
wondered if overexpression of XOtx5b increased the proportion of
rods, cones, or both. To address this question, we used the cone
photoreceptor-specific anti-calbindin antibody to identify cones in
XOtx5b lipofected retinas. To increase the number of photoreceptors
in this analysis, we used XOtx5bN+HD-VP16 for our in vivo
lipofections (see below for further details of this construct).
XOtx5bN+HD-VP16 dramatically increased the proportion of
photoreceptor cells from 27% to 71% (Fig.
5C). When we stained these same sections for the cone marker,
calbindin, we found approximately equal numbers of calbindin-positive and
-negative photoreceptors (Fig.
5D). In control, GFP and vector only lipofected retinas,
we also found approximately equal numbers of rods and cones
(Fig. 5D), which is consistent
with previous reports (Chang and Harris,
1998). Together these results demonstrate that
XOtx5bN+HD-VP16 increases the proportion of both rods and cones in
equal numbers.
|
XOtx5b and XOtx2 N-terminal and HD fused to the engrailed repressor
suggest that XOtx5b and XOtx2 have some cell fate specification
activities
Transcription factors can act as activators, repressors, or both. Mouse Crx
has been shown to activate rhodopsin but repress NeuroD,
either directly or through a cascade of other factors
(Furukawa et al., 1999;
Livesey et al., 2000
). We were
interested to know if XOtx5b and XOtx2 worked primarily as
repressors or activators when modulating retinal cell fates. Therefore, we
fused the powerful repressor domain of engrailed to the N terminus
and homeodomain (HD) of XOtx5b (XOtx5bN+HD-EngR) and XOtx2
(XOtx2N+HD-EngR) and determined the effect of these fusions on cell fate
(Fig. 5E-G). Lipofection of the
XOtx5bN+HD-EngR led to fewer lipofected photoreceptor cells than
controls (P=0.001; Fig.
5G). This suggests activation of XOtx5b target genes
stimulates transcription necessary for photoreceptor cell specification.
XOtx2N+HD-EngR lipofected retinas showed a decrease in the percentage
of lipofected bipolar cells compared to controls (P=0.004;
Fig. 5G), suggesting that
native XOtx2 also acts as an activator of genes necessary for bipolar
cell specification. These results also suggest some control of cell fate
specification by the N-terminal and HD sequences.
Engrailed fusion constructs of XOtx5b and XOtx2 also gave some unexpected results. XOtx5bN+HD-EngR did not affect ganglion, amacrine or Müller cell populations, while the proportion of bipolar cells was slightly increased (5%, P<0.05). XOtx2N+HD-EngR increased the proportion of ganglion cells (by 4%, P<0.02) but did not alter the photoreceptor cell population. These results demonstrate that although full-length XOtx2 and XOtx5b may modulate photoreceptor levels, they both may act in more complex ways than as dedicated repressors or activators.
These results also suggest that the N-terminal and HD regions of these
proteins are not sufficient to direct cell fate specification in the same way
as the full-length proteins. This fits with a recent study that compares
otd/Otx family members (Sauka-Spengler et
al., 2001) and the fact that Xenopus XOtx5b and XOtx2
have very similar homeodomains and N-terminal regions. Indeed,
Xenopus XOtx5b and XOtx2 homeodomains (HD) have only one residue
difference (XOtx2=A; XOtx5b=S) while the region N-terminal
to the HD, which we called the N terminus, is 73% identical in these two
proteins. If the N terminus and homeodomain were mostly responsible for the
differences we observed in the lipofection results between these two
full-length constructs, and if, as the above results suggest, that some
aspects of the phenotype were due to their activity as activators rather than
repressors, then a construct which fuses the N terminus and HD to the strong
activator VP16 should mimic a full-length construct. To test this, we fused
the N terminus and HD to the strong activator domain of VP16 for each of these
genes (XOtx2N+HD-VP16 and XOtx5bN+HD-VP16) and lipofected
each with the tracer GFP into stage 18 embryos
(Fig. 5H,I). In each case, the
number of GFP-positive photoreceptor cells increased by about three
times that of the control lipofected retinas (both P<0.0001) at
the expense of all the other retinal cell types
(Fig. 5J). These results
suggest that the N terminus and homeodomain may be involved in regulating
photoreceptor cell specification but show a striking lack of specificity
between photoreceptors and bipolars. The increased percentage of photoreceptor
cells in retinas lipofected with the VP16 activator fusion constructs
(XOtx5bN+HD-VP16=80% and GFP=27%) was even more than retinas
lipofected with full-length XOtx5b (XOtx5b=37% and GFP=28%;
Fig. 4D compared with
Fig. 5J). We therefore had to
consider the possibility that the VP16 activator alone may be responsible for
photoreceptor cell fate. To test this, we injected a VP16 activator
construct fused to a nuclear localization sequence and GFP into
developing embryos and found no effect on cell fate (data not shown). It could
also be that the N terminus and homeodomain can activate
photoreceptor-specific genes on their own and adding the VP16
activator domain made the constructs even stronger transactivators. Hence, we
lipofected retinas with just the N terminus and homeodomain of either XOtx5b
or XOtx2, but in neither case was there an effect on cell fate (data not
shown). These results suggest that the N-terminal and HD of both XOtx5b and
XOtx2 may have a common function in retinal cell determination, and be part of
the shared regulatory mechanisms of photoreceptors and bipolars
(Chen et al., 1994
;
Chiu and Nathans, 1994
). The
lack cell type specificity of these domains when fused to an activator
suggested that the region C-terminal to the HD may be more important in
modulating cell fate.
The C-terminus of either XOtx2 or XOtx5b is involved in photoreceptor
versus bipolar fate determination
C-terminal to the homeodomain, otd/Otx family member genes have several
unique domains: a glutamine (Gln) rich region, a WSP domain and an OTX tail
sequence (review by Morrow et al.,
1998). Comparison of the entire XOtx2 and XOtx5b C termini
sequence shows that they are 60.1% identical (data not shown). To test if the
difference we observed in the way XOtx2 and XOtx5b
overexpression affects retinal cell fate maps to the C terminus, we did domain
swap experiments. The N terminus and HD of XOtx2 was fused to the C
terminus of XOtx5b (XOtx2/5b) and the N-terminal and HD
sequence of XOtx5b was fused to the C terminus of XOtx2
(XOtx5b/2; Fig. 6A). Each domain swapped construct was then co-lipofected with GFP into
developing embryos as above (Fig.
6C,D) and the ratios of each cell type calculated
(Fig. 6E,F). XOtx5b/2,
like XOtx2 itself, increased bipolar cells (P=0.0001) at the
expense of photoreceptor (P=0.031) and Müller
(P=0.0001) cells while XOtx2/5b, like XOtx5b
itself, increased photoreceptor cells (P=0.0004) at the expense of
ganglion (P=0.004), amacrine (P=0.018), Müller
(P=0.005) and horizontal (P=0.021) cells. These results
clearly suggest that the C-terminal region of XOtx2 and
XOtx5b is much more critical for cell fate specification affects than
the N terminus and HD.
|
XOtx2 and XOtx5b transcripts are found in
Brd-positive cells but do not affect retinal proliferation or apoptosis
Increases in photoreceptor or bipolar cell percentages in the population of
XOtx5b or XOtx2 lipofected cells, respectively, could be due
cell type-specific changes in proliferation or death. Previous studies have
shown that XOtx2 is expressed in proliferating retinoblasts
(Perron et al., 1998) but
XOtx5b expression in dividing cells has not been described. To test
if XOtx5b is normally expressed in mitotic cells, we injected embryos
with BrdU and fixed them 30 minutes later. We sectioned their retinas and
stained for XOtx5b or XOtx2 expression by in situ
hybridisation, and afterwards, for BrdU by immunostaining to look for
XOtx5b- or XOtx2-positive cells that were also BrdU positive
(Fig. 7A-F). We found that most
of the cells that expressed XOtx5b or XOtx2 were not BrdU
positive. However, a few cells in the CMZ that were XOtx5b or
XOtx2 positive, were also labelled with BrdU. This confirmed that
XOtx2 is in some proliferating cells and indicated that
XOtx5b can also be found in proliferating cells of the retina, as
suggested by their expression during early stages of retinal development.
|
As these transcripts are present in BrdU-positive cells, it is possible that they cause an increase in proliferation of the respective cell types they influence. To test this, we lipofected developing retinas with GFP and each one of the following constructs in separate experiments: XOtx5b, XOtx5bN+HD-VP16, XOtx5bN+HD-EngR, XOtx2, XOtx2N+HD-VP16 and the swapped domain constructs (XOtx2/5b and XOtx5b/2) at stage 18. At stage 31 (the beginning of photoreceptor cell differentiation), we injected the embryos with BrdU. At stage 41 (retinal maturation), the embryos were fixed and the retinas sectioned and stained for GFP and BrdU. Cells expressing just GFP or GFP and BrdU were counted. We found that none of the constructs we tested showed an increase in proliferation in any of the cell types at this stage, suggesting that these constructs do not influence proliferation (Fig. 7G,H).
To produce an increase in the percentage of lipofected bipolar cells, XOtx2 could cause cell death in other retinal cell types, like photoreceptors or Müller cells. Similarly, XOtx5b lipofected retinal cells that are not photoreceptors might be more likely to undergo programmed cell death. To test if either of these constructs caused apoptosis, we lipofected developing retinas as above, fixed at stages 31, 33/34 and 37 and stained the sections for apoptotic nuclei using a TUNEL assay. The number of GFP-positive cells in each of the retinas was counted as well as those apoptotic cells that were GFP positive. In each case, the percentage of GFP-positive cells that were also apoptotic was around 2-4%. Statistical analysis using a Student's t-test showed no differences (data not shown). This suggests that selective cell death is also not the basis for the overexpression phenotypes. The most likely explanation for the phenotypes observed, therefore, is that these constructs cause a fate switch or conversion.
Xopsin reporter is activated by XOtx5b and this activation
is suppressed by XOtx2
Crx binds to the OTX-binding consensus sequence found in the rhodopsin
promoter and transactivates its expression
(Chen et al., 1997;
Furukawa et al., 1997
). Like
Crx, Otx2 also binds this sequence and has been shown to both activate and
repress genes by binding to this site
(Kelley et al., 2000
;
Morgan et al., 1999
). Since
XOtx5b has been suggested to be part of the same subfamily of otd/Otx
transcription factors as Crx (Germot et
al., 2001
; Sauka-Spengler et
al., 2001
), we reasoned that XOtx5b might also regulate
opsin expression. To test this hypothesis, we injected one dorsal
cell of a 4-cell stage blastomere with an expression vector of GFP driven by
the Xenopus opsin promoter [Xop-GFP
(Mani et al., 2001
)], capped
XOtx5b RNA and ß-gal RNA. We allowed the embryos to grow to
stage 17-18, they were then fixed and the fluorescence quantified. As a
negative control, Xop-GFP DNA and ß-gal RNA were injected alone
(Fig. 7I). All embryos that
were injected with Xop-GFP DNA and XOtx5b RNA on the dorsal side
(Fig. 7J), expressed GFP at
levels higher than the negative control embryos
(Fig. 7I,
P<0.0001), suggesting that XOtx5b does activate the opsin
promoter.
Since XOtx5b is expressed in a subset of bipolar cells with
XOtx2, we were interested to know whether XOtx2 affected the
ability of XOtx5b to regulate opsin expression. Co-injection of equal
amounts of XOtx2 and XOtx5b RNA plus Xop-GFP DNA and
ß-gal RNA (Fig. 7K) showed
that XOtx2 buffers the effect of XOtx5b on Xop-GFP; i.e.
levels of GFP expression are similar to those of the uninjected side
(Fig. 7N). Staining for
ß-gal confirmed that these embryos were successfully injected with the
RNA/DNA mixture. By adding double the concentration of XOtx2 than
XOtx5b RNA plus Xop-GFP DNA and ß-gal RNA
(Fig. 7L), we still observed a
suppression of the XOtx5b-driven opsin activation. However,
when we added the Xop-GFP DNA and ß-gal and XOtx2 RNA alone
(Fig. 7M), we found that
opsin expression was enhanced by XOtx2. This was not
surprising since either Crx and Otx2 alone activates the
promoter of another photoreceptor-specific gene, interphotoreceptor retinoid
binding protein (IRBP) (Bobola et al.,
1999). Our results with the Xenopus opsin promoter
suggest that the relative levels of XOtx2 and XOtx5b are
important in determining the activation or repression of opsin. It
appears that XOtx2 plays a role in suppressing the effect of
XOtx5b on opsin expression when equal or double the amounts
of XOtx2 to XOtx5b are present in the cell.
XOtx2 suppresses the XOtx5b enhancement of
photoreceptor cell fate
The XOtx2 suppression of the activation of a
photoreceptor-specific gene in the above assay may partly explain why bipolar
cells do not normally express opsins. A similar question raised by this study
is why cells that express both XOtx5b and XOtx2 appear to
differentiate into bipolars rather than photoreceptors. Could it be that
XOtx2 suppresses the ability of XOtx5b to induce
photoreceptors? To test this hypothesis, we colipofected XOtx2 and
XOtx5b with GFP into the eye field of stage 18 embryos as described
above. Controls were lipofected with XOtx5b and GFP or GFP and vector
alone. XOtx5b-lipofected retinas had the expected increase in
photoreceptor cells (P=0.001). However, when XOtx2 and
XOtx5b were colipofected, there was no increase in photoreceptors but
rather an increase in the population of lipofected bipolar cells
(Fig. 7O-R; P=0.007).
These results suggest that when XOtx2 and XOtx5b are both
present in progenitors, cells are more likely to take on bipolar cell
fates.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Contrary to mammalian and zebrafish Crx studies
(Chen et al., 1997;
Furukawa et al., 1997
;
Liu et al., 2001
), Xenopus
XOtx5b is expressed at much earlier stages of development and plays a
role similar to XOtx2 in early embryogenesis
(Vignali et al., 2000
).
XOtx5b is expressed in dividing retinal cells and in all layers of
the undifferentiated retina during development, which is similar to zebrafish
Crx, but unlike mammalian Crx, which is expressed in
photoreceptor cells just after they are born
(Chen et al., 1997
;
Furukawa et al., 1997
;
Liu et al., 2001
;
Morrow et al., 1998
). We also
found that XOtx5b-engrailed constructs resulted in clones with a
decrease in photoreceptor cells. This is also different from the
Crx-engrailed repressor fusion construct results, which produced
clones with rods lacking outer segments
(Furukawa et al., 1997
). The
ability of XOtx5b to decrease the number of photoreceptors, albeit a
small amount in comparison to the activation seen by the VP16 activator fusion
constructs, suggests that XOtx5b may be playing an earlier role in
photoreceptor specification than Crx. Phylogenetic analysis suggests
that XOtx5b and Crx may be orthologous
(Germot et al., 2001
;
Sauka-Spengler et al., 2001
)
suggesting that one might consider renaming Xenopus Otx5b, XCrx. Our
findings that XOtx5b functions similarly to mammalian Crx,
supports this idea. However, since there are clear differences between the
roles played by mammalian Crx and Xenopus XOtx5b before eye
formation, and since it is still possible that a Xenopus Crx gene
with more sequence similarity exists as was found in teleosts, we would
suggest that Xenopus XOtx5b is not renamed.
To date, zebrafish is the only species reported to have separate genes
encoding Crx and Otx5 (Gamse et al., 2002). Xenopus also has two
genes in the hypothesised Otx5/Crx orthology class, XOtx5
(Kuroda et al., 2000) and
XOtx5b (Vignali et al.,
2000
). Although zebrafish Crx and Otx5 retinal
and pineal expression are the same, their protein sequences are only 64%
identical (data not shown) and they regulate pineal circadian gene expression
differently (Gamse et al., 2002). This suggests that they are different genes.
Although XOtx5 retinal expression has not yet been described, the
early embryonic XOtx5 and XOtx5b expression patterns are
virtually identical (Kuroda et al.,
2000
; Vignali et al.,
2000
) and comparison of their protein sequences show they are 96%
identical (data not shown). Injections of the blastomere with either
XOtx5 or XOtx5b results in embryos of reduced size with
posterior deficiencies and ectopic cement gland structures
(Kuroda et al., 2000
;
Vignali et al., 2000
),
suggesting that the two genes function similarly in the embryo. Given the
pseudotetrapoid Xenopus laevis genome, these results strongly suggest
that XOtx5 and XOtx5b arose from a gene duplication event.
This is further supported by phylogenetic analysis unambiguously placing
XOtx5 and XOtx5b in the same orthology class
(Germot et al., 2001
;
Sauka-Spengler et al.,
2001
).
XOtx2 promotes bipolar cell specification
Previously, XOtx2 has been shown to be involved in anteriorizing
the embryo and to have a role in cement gland formation
(Andreazzoli et al., 1997;
Blitz and Cho, 1995
;
Gammill and Sive, 1997
;
Gammill and Sive, 2001
;
Pannese et al., 1995
). Here,
we show that it is involved in specifying the bipolar cell. Recent studies on
bipolar cell specification have examined several transcription factors using
transgenic and mutant mice. Mice carrying a null mutation in Chx10
produce retinas lacking differentiated bipolar cells
(Burmeister et al., 1996
).
Similarly, Mash1-Math3 double knockout mice retinas are also missing
bipolar cells, yet neither mutation alone affects bipolar cell production
(Tomita et al., 2000
;
Tomita et al., 1996
). Viral
infection experiments show that together, Chx10, Mash1 and
Math3, are all involved in bipolar cell genesis
(Hatakeyama et al., 2001
).
However, not all cells that co-expressed Chx10 and either
Mash1 or Math3 become bipolars, suggesting that other
factors are also necessary. Like Chx10, XOtx2 is expressed throughout
the undifferentiated neural retina and later is restricted to differentiated
bipolar cells. Like Chx10 and either Mash1 or Math3
overexpression (Hatakeyama et al.,
2001
), overexpression of XOtx2 in developing retinal
cells increases the number of bipolar cells in that population. Consistent
with this, we found that lipofection of the N terminus and DNA binding domain
of XOtx2 fused to the engrailed transcription repressor
decreases the percentage of lipofected bipolar cells, suggesting that
XOtx2 acts as an activator to generate an increase in lipofected
bipolar cells. It will be interesting to know if these four genes together,
XOtx2, Chx10, Mash1 and Math3, are sufficient for directing
all retinoblasts to a bipolar cell fate.
The C terminus of both XOtx2 and XOtx5b are necessary for their cell
fate specific activities
Analysis of deletion constructs of Crx and their ability to
transactivate the bovine rhodopsin promoter lead to the conclusion that
regions C-terminal to the homeodomain are important for gene activation
(Chau et al., 2000). Moreover,
they showed that the N terminus, homeodomain and basic region (aa 1-107) are
necessary for binding the BAT-1 site on the rhodopsin promoter but are poor
transactivators of rhodopsin (Chau et al.,
2000
). Consistent with this, we found that lipofection of this
same region of XOtx2 (aa 1-109) or XOtx5b (aa 1-107) does
not have any effect on photoreceptor cell fate, but when fused to the
engrailed repressor or VP16 activator, lipofection of these fusion
constructs influences the cell fate of the lipofected cells. Even though this
region (aa 1-107) can successfully target the rhodopsin promoter in vitro
(Chau et al., 2000
), it may be
that removal of such a large area of the protein (aa 110-289 for XOtx2; aa
108-290 for XOtx5b) could alter the ability of the fusion constructs to
correctly target DNA cis-acting elements in vivo. However, when we
swapped the C-terminal region of these proteins onto the opposite N-terminal
and homeodomain sequence, we found that the region C-terminal to the
homeodomain of XOtx5b and XOtx2 are capable of producing the
same phenotype as XOtx5b and XOtx2, respectively,
independently of which N terminus and HD they are fused to.
Clearly, the region C-terminal to the homeodomain of XOtx5b and
XOtx2 is not simply required for activation or repression of target
genes, as overexpression of the DNA binding domain and the sequence N-terminal
to the homeodomain of either XOtx5b (aa 1-107) or XOtx2 (aa
1-109) fused to a VP16 activator or engrailed repressor produced a
different labelled retinal cell distribution than overexpression with either
the XOtx2 or XOtx5b alone. It has been shown that XOtx2
directly activates some genes like that of the Xenopus transcription
factor Clock (Green et al.,
2001), and indirectly activates the secreted protein, XAG, while
it directly represses others, like the transcription factor Brachyury and the
secreted factor, Wnt-5a (Latinkic et al.,
1997
; Morgan et al.,
1999
) (reviewed by Boncinelli
and Morgan, 2001
).
A basic leucine zipper protein, NRL, has been shown to work synergistically
with Crx to transactivate the bovine rhodopsin promoter
(Chen et al., 1997). Studies
done on the binding of Crx with NRL suggest that the basic domain and
homeodomain of Crx are necessary for this interaction
(Mitton et al., 2000
).
Deletion analysis on Crx shows that the region C-terminal to the homeodomain
is necessary for photoreceptor-specific gene activation with or without NRL.
The fact that the VP16 activator fused to either XOtx2 or
XOtx5b N-terminal region and homeodomain induces photoreceptor cells,
suggest that these regions may be necessary for targeting genes involved in
photoreceptor specification even if they are not sufficient to specify cell
fate effectively. Deletion analysis also revealed that half of the
transactivation function of Crx was lost upon removal of the Otx tail
(Chau et al., 2000
).
Comparison of the Otx tail of all the Crx sequences listed to eight different
species of Otx2 homologues showed an extra threonine (T) in the second repeat
of the Otx tail of all the Otx2 proteins. Thus, site directed mutation
analysis may clarify which residues in the C terminus of XOtx2 and XOtx5b are
critical for the unique function of each of these closely related family
members.
Photoreceptors versus bipolars, Otx5b versus
Otx2
How are XOtx2 and XOtx5b involved in retinal cell fate
specification? Our results confirm other studies done on chick
(Bovolenta et al., 1997) that
early in retina formation, XOtx2 transcripts are found in all layers
of the neural retina. Yet, if XOtx2 is involved in bipolar cell
specification in Xenopus, why do not all cells become bipolar cells?
Similarly, XOtx5b is expressed in dividing retinoblasts in all layers
of the early retina during its formation but is involved in specification of
photoreceptor cells. This suggests that different transcription factors may be
competing or cooperating in cell fate specification in a combinatorial way.
The results with the Xenopus rhodopsin promoter may shed light on
this. Both XOtx5b and XOtx2 alone can activate opsin
expression but together they block activation of opsin. A similar mechanism
has been shown for Barrier-to-autointegration-factor (Baf), which is expressed
in bipolars with Crx in the mammalian retina. Baf interacts directly with Crx
to repress Crx-mediated transactivation of a rhodopsin reporter
(Wang et al., 2002
). A
possible mechanism for how the Otx transcription factors may interact to
produce a similar result comes from recent studies of mouse Otx3, which is
expressed in the developing eye, binds to the same consensus binding sequence
as Crx and yet appears to act as a repressor rather than an activator
(Zhang et al., 2002
). This
idea of cooperative specification is supported by the results of the
co-lipofection experiment showing that misexpressed XOtx5b on its own
induces photoreceptors but the combination of the XOtx2 and
XOtx5b overrides this activity and induces bipolar cells. We hope
that these findings may stimulate insight or speculation into the
developmental and evolutionary mechanisms whereby overlapping patterns of
homologous genes within a tissue specify the fates of similar and possibly
homologous cell types within that tissue, much as Hox genes specify segmetal
identity at the tissue level.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acampora, D., Avantaggiato, V., Tuorto, F., Barone, P.,
Reichert, H., Finkelstein, R. and Simeone, A. (1998a). Murine
Otx1 and Drosophila otd genes share conserved genetic functions required in
invertebrate and vertebrate brain development.
Development 125,1691
-1702.
Acampora, D., Avantaggiato, V., Tuorto, F., Briata, P., Corte,
G. and Simeone, A. (1998b). Visceral endoderm-restricted
translation of Otx1 mediates recovery of Otx2 requirements for specification
of anterior neural plate and normal gastrulation.
Development 125,5091
-5104.
Acampora, D., Mazan, S., Avantaggiato, V., Barone, P., Tuorto, F., Lallemand, Y., Brulet, P. and Simeone, A. (1996). Epilepsy and brain abnormalities in mice lacking the Otx1 gene. Nat. Genet. 14,218 -222.[Medline]
Acampora, D. and Simeone, A. (1999). The TINS Lecture. Understanding the roles of Otx1 and Otx2 in the control of brain morphogenesis. Trends Neurosci. 22,116 -122.[CrossRef][Medline]
Altshuler, D. and Cepko, C. (1992). A temporally regulated, diffusible activity is required for rod photoreceptor development in vitro. Development 114,947 -957.[Abstract]
Andreazzoli, M., Pannese, M. and Boncinelli, E.
(1997). Activating and repressing signals in head development:
the role of Xotx1 and Xotx2. Development
124,1733
-1743.
Baas, D., Bumsted, K. M., Martinez, J. A., Vaccarino, F. M., Wikler, K. C. and Barnstable, C. J. (2000). The subcellular localization of OTX2 is cell-type specific and developmentally regulated in the mouse retina. Brain Res. Mol. Brain Res. 78, 26-37.[Medline]
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.
Belliveau, M. J., Young, T. L. and Cepko, C. L.
(2000). Late retinal progenitor cells show intrinsic limitations
in the production of cell types and the kinetics of opsin synthesis.
J. Neurosci. 20,2247
-2254.
Blitz, I. L. and Cho, K. W. (1995). Anterior
neurectoderm is progressively induced during gastrulation: the role of the
Xenopus homeobox gene orthodenticle.
Development 121,993
-1004.
Bobola, N., Briata, P., Ilengo, C., Rosatto, N., Craft, C., Corte, G. and Ravazzolo, R. (1999). OTX2 homeodomain protein binds a DNA element necessary for interphotoreceptor retinoid binding protein gene expression. Mech. Dev. 82,165 -169.[CrossRef][Medline]
Boncinelli, E. and Morgan, R. (2001). Downstream of Otx2, or how to get a head. Trends Genet 17,633 -636.[CrossRef][Medline]
Bovolenta, P., Mallamaci, A., Briata, P., Corte, G. and
Boncinelli, E. (1997). Implication of OTX2 in pigment
epithelium determination and neural retina differentiation. J.
Neurosci. 17,4243
-4252.
Broadbent, J. and Read, E. M. (1999). Wholemount in situ hybridization of Xenopus and zebrafish embryos. In Methods in Molecular Biology: Molecular Methods in Developmental Biology Xenopus and Zebrafish (ed. M. Guille), pp.58 -61. Totowa, NJ: Humana Press.
Burmeister, M., Novak, J., Liang, M. Y., Basu, S., Ploder, L., Hawes, N. L., Vidgen, D., Hoover, F., Goldman, D., Kalnins, V. I. et al. (1996). Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation. Nat. Genet. 12,376 -384.[Medline]
Carter-Dawson, L. D. and LaVail, M. M. (1979). Rods and cones in the mouse retina. II. Autoradiographic analysis of cell generation using tritiated thymidine. J. Comp. Neurol. 188,263 -272.[Medline]
Chang, W. S. and Harris, W. A. (1998). Sequential genesis and determination of cone and rod photoreceptors in Xenopus. J. Neurobiol. 35,227 -244.[CrossRef][Medline]
Chau, K., Chen, S., Zack, D. J. and Ono, S. J.
(2000). Functional domains of the cone-rod homeobox (Crx)
transcription factor. J. Biol. Chem.
275,37264
-37270.
Chen, J., Tucker, C. L., Woodford, B., Szel, A., Lem, J., Gianella-Borradori, A., Simon, M. I. and Bogenmann, E. (1994). The human blue opsin promoter directs transgene expression in short- wave cones and bipolar cells in the mouse retina. Proc. Natl. Acad. Sci. USA 91,2611 -2615.[Abstract]
Chen, S., McMahan, B. and Xu, S. (2000). Localization of the Crx protein in the mature and developing mouse retina. Assoc. Res. Vis. Ophthalmol. 41, 5392.
Chen, S., Wang, Q. L., Nie, Z., Sun, H., Lennon, G., Copeland, N. G., Gilbert, D. J., Jenkins, N. A. and Zack, D. J. (1997). Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron 19,1017 -1030.[Medline]
Chiu, M. I. and Nathans, J. (1994). Blue cones and cone bipolar cells share transcriptional specificity as determined by expression of human blue visual pigment-derived. J. Neurosci. 14,3426 -3436.[Abstract]
Ezzeddine, Z. D., Yang, X., DeChiara, T., Yancopoulos, G. and
Cepko, C. L. (1997). Postmitotic cells fated to become rod
photoreceptors can be respecified by CNTF treatment of the retina.
Development 124,1055
-1067.
Fields-Berry, S. C., Halliday, A. L. and Cepko, C. L. (1992). A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina. Proc. Natl. Acad. Sci. USA 89,693 -697.[Abstract]
Finkelstein, R., Smouse, D., Capaci, T. M., Spradling, A. C. and Perrimon, N. (1990). The orthodenticle gene encodes a novel homeo domain protein involved in the development of the Drosophila nervous system and ocellar visual structures. Genes Dev. 4,1516 -1527.[Abstract]
Furukawa, T., Morrow, E. M. and Cepko, C. L. (1997). Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell 91,531 -541.[Medline]
Furukawa, T., Morrow, E. M., Li, T., Davis, F. C. and Cepko, C. L. (1999). Retinopathy and attenuated circadian entrainment in Crx-deficient mice. Nat. Genet. 23,466 -470.[CrossRef][Medline]
Gammill, L. S. and Sive, H. (1997).
Identification of otx2 target genes and restrictions in ectodermal competence
during Xenopus cement gland formation.
Development 124,471
-481.
Gammill, L. S. and Sive, H. (2001). otx2 expression in the ectoderm activates anterior neural determination and is required for Xenopus cement gland formation. Dev. Biol. 240,223 -236.[CrossRef][Medline]
Gamse, J. T., Shen, Y. C., Thisse, C., Thisse, B., Raymond, P. A., Halpern, M. E. and Liang, J. O. (2001). Otx5 regulates genes that show circadian expression in the zebrafish pineal complex. Nat. Genet. 30,117 -121.[CrossRef]
Gawantka, V., Pollet, N., Delius, H., Vingron, M., Pfister, R., Nitsch, R., Blumenstock, C. and Niehrs, C. (1998). Gene expression screening in Xenopus identifies molecular pathways, predicts gene function and provides a global view of embryonic patterning. Mech. Dev. 77,95 -141.[CrossRef][Medline]
Germot, A., Lecointre, G., Plouhinec, J. L., le Mentec, C.,
Girardot, F. and Mazan, S. (2001). Structural evolution of
Otx genes in craniates. Mol. Biol. Evol.
18,1668
-1678.
Green, C. B., Durston, A. J. and Morgan, R. (2001). The circadian gene Clock is restricted to the anterior neural plate early in development and is regulated by the neural inducer noggin and the transcription factor Otx2. Mech. Dev. 101,105 -110.[CrossRef][Medline]
Greferath, U., Grunert, U. and Wassle, H. (1990). Rod bipolar cells in the mammalian retina show protein kinase C-like immunoreactivity. J. Comp. Neurol. 301,433 -442.[Medline]
Hatakeyama, J., Tomita, K., Inoue, T. and Kageyama, R.
(2001). Roles of homeobox and bHLH genes in specification of a
retinal cell type. Development
128,1313
-1322.
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]
Kablar, B., Vignali, R., Menotti, L., Pannese, M., Andreazzoli, M., Polo, C., Giribaldi, M. G., Boncinelli, E. and Barsacchi, G. (1996). Xotx genes in the developing brain of Xenopus laevis. Mech. Dev. 55,145 -158.[CrossRef][Medline]
Kelley, C. G., Lavorgna, G., Clark, M. E., Boncinelli, E. and
Mellon, P. L. (2000). The Otx2 homeoprotein regulates
expression from the gonadotropin-releasing hormone proximal promoter.
Mol. Endocrinol. 14,1246
-1256.
Kuroda, H., Hayata, T., Eisaki, A. and Asashima, M. (2000) Cloning a novel developmental regulating gene, Xotx5: its potential role in anterior formation in Xenopus laevis. Dev. Growth Differ. 42,87 -93.[CrossRef][Medline]
Latinkic, B. V., Umbhauer, M., Neal, K. A., Lerchner, W., Smith,
J. C. and Cunliffe, V. (1997). The Xenopus Brachyury promoter
is activated by FGF and low concentrations of activin and suppressed by high
concentrations of activin and by paired-type homeodomain proteins.
Genes Dev. 11,3265
-3276.
Liu, Y., Shen, Y. C., Rest, J. S., Raymond, P. A. and Zack, D.
J. (2001). Isolation and characterization of a zebrafish
Hhomologue of the cone rod homeobox gene. Invest. Ophthalmol. Vis.
Sci. 42,481
-487.
Livesey, F. J. and Cepko, C. L. (2001). Vertebrate neural cell-fate determination: lessons from the retina. Nat. Rev. Neurosci. 2,109 -118.[CrossRef][Medline]
Livesey, F. J., Furukawa, T., Steffen, M. A., Church, G. M. and Cepko, C. L. (2000). Microarray analysis of the transcriptional network controlled by the photoreceptor homeobox gene Crx. Curr. Biol. 10,301 -310.[CrossRef][Medline]
Mani, S. S., Batni, S., Whitaker, L., Chen, S., Engbretson, G.
and Knox, B. E. (2001). Xenopus rhodopsin promoter.
Identification of immediate upstream sequences necessary for high level,
rod-specific transcription. J. Biol. Chem.
276,36557
-36565.
Martinez-Morales, J. R., Signore, M., Acampora, D., Simeone, A.
and Bovolenta, P. (2001). Otx genes are required for tissue
specification in the developing eye. Development
128,2019
-2030.
Mitton, K. P., Swain, P. K., Chen, S., Xu, S., Zack, D. J. and
Swaroop, A. (2000). The leucine zipper of NRL interacts with
the CRX homeodomain. J. Biol. Chem.
275,29794
-29799.
Morgan, R., Hooiveld, M. H., In der Reiden, P. and Durston, A. J. (1999). A conserved 30 base pair element in the Wnt-5a promoter is sufficient both to drive its early embryonic expression and to mediate its repression by otx2. Mech. Dev. 85, 97-102.[CrossRef][Medline]
Morrow, E. M., Furukawa, T. and Cepko, C. L. (1998). Vertebrate photoreceptor cell development and disease. Trends Cell Biol. 8,353 -358.[CrossRef][Medline]
Myat, A., Henrique, D., Ish-Horowicz, D. and Lewis, J. (1996). A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogenesis. Dev. Biol. 174,233 -247.[CrossRef][Medline]
Pannese, M., Polo, C., Andreazzoli, M., Vignali, R., Kablar, B.,
Barsacchi, G. and Boncinelli, E. (1995). The Xenopus
homologue of Otx2 is a maternal homeobox gene that demarcates and specifies
anterior body regions. Development
121,707
-720.
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]
Sauka-Spengler, T., Barratte, B., Shi, D. L. and Mazan, S. (2001). Structure and expression of an Otx5-related gene in the dogfish Scyliorhinus canicula: evidence for a conserved role of Otx5 and Crx genes in the specification of photoreceptors. Dev. Genes Evol. 211,533 -544.[CrossRef][Medline]
Shimamura, K., Hirano, S., McMahon, A. P. and Takeichi, M.
(1994). Wnt1-dependent regulation of local E-cadherin and alpha
N-catenin expression in the embryonic mouse brain.
Development 120,2225
-2234.
Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A. and Boncinelli, E. (1992). Nested expression domains of four homeobox genes in developing rostral brain. Nature 358,687 -690.[CrossRef][Medline]
Stiemke, M. M. and Hollyfield, J. G. (1995). Cell birthdays in Xenopus laevis retina. Differentiation 58,189 -193.[CrossRef][Medline]
Suda, Y., Nakabayashi, J., Matsuo, I. and Aizawa, S.
(1999). Functional equivalency between Otx2 and Otx1 in
development of the rostral head. Development
126,743
-757.
Tomita, K., Moriyoshi, K., Nakanishi, S., Guillemot, F. and
Kageyama, R. (2000). Mammalian achaete-scute and atonal
homologs regulate neuronal versus glial fate determination in the central
nervous system. EMBO J.
19,5460
-5472.
Tomita, K., Nakanishi, S., Guillemot, F. and Kageyama, R.
(1996). Mash1 promotes neuronal differentiation in the retina.
Genes Cells 1,765
-774.
Turner, D. L. and Cepko, C. L. (1987). A common progenitor for neurons and glia persists in rat retina late in development. Nature 328,131 -136.[CrossRef][Medline]
Turner, D. L., Snyder, E. Y. and Cepko, C. L. (1990). Lineage-independent determination of cell type in the embryonic mouse retina. Neuron 4, 833-845.[Medline]
Vignali, R., Colombetti, S., Lupo, G., Zhang, W., Stachel, S., Harland, R. M. and Barsacchi, G. (2000). Xotx5b, a new member of the otx gene family, may be involved in anterior and eye development in xenopus laevis. Mech. Dev. 96, 3-13.[CrossRef][Medline]
Wang, X., Xu, S., Rivolta, C., Li, L. Y., Peng, G. H., Swain, P.
K., Sung, C. H., Swaroop, A., Berson, E. L. et al. (2002).
Barrier-to-autointegration factor interacts with the cone-rod homeobox and
represses its transactivation function. J. Biol. Chem.
277,43288
-43300.
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]
Young, R. W. (1985). Cell differentiation in the retina of the mouse. Anat. Rec. 212,199 -205.[Medline]
Zhang, Y., Miki, T., Iwanaga, T., Koseki, Y., Okuno, M., Sunaga,
Y., Ozaki, N., Yano, H., Koseki, H. and Seino, S. (2002).
Identification, tissue expression, and functional characterization of Otx3, a
novel member of the Otx family. J. Biol. Chem.
277,28065
-28069.
Zuber, M. E., Perron, M., Philpott, A., Bang, A. and Harris, W. A. (1999). Giant eyes in Xenopus laevis by overexpression of XOptx2. Cell 98,341 -352.[Medline]