1 Program in Developmental Biology, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030, USA
2 Departments of Molecular and Cellular Biology, Baylor College of Medicine, One
Baylor Plaza, Houston, TX 77030, USA
3 Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
4 Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
5 Department of Biology, Ithaca College, Ithaca, NY 14850, USA
Author for correspondence (e-mail:
jamrich{at}bcm.tmc.edu)
Accepted 27 May 2003
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SUMMARY |
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Key words: FGF receptor, Photoreceptors, Retina, Transgenic, Xenopus, Xrx1A
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INTRODUCTION |
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As Rx shows specific expression in the retinal progenitor cells, its regulatory sequences would be uniquely suited to direct gene expression in the developing retina. These sequences could be used to specifically alter gene expression in the developing eye. In this study, we used a transgenic approach in Xenopus laevis to identify the regulatory sequences of the Xrx1A gene. Having identified these sequences, we used them to demonstrate the crucial role of FGF signaling in the correct specification of retinal cell types.
Fibroblast Growth Factors (FGFs) are a family of signaling molecules that are expressed in a wide range of tissues in partially overlapping patterns. FGFs have been implicated to have roles in the formation of mesoderm and neuroectoderm. Their exact role during embryogenesis is not fully understood, partially because of several contradictory findings.
In Xenopus there are at least five FGF receptors
(Friesel and Brown, 1992;
Gillespie et al., 1989
;
Golub et al., 2000
;
Hongo et al., 1999
;
Shiozaki et al., 1995
) and it
would not be surprising if additional members of this gene family are
discovered in the future. The dominant-negative forms of these receptors have
proved to be efficient inhibitors of FGF signaling pathways and have been used
as the primary means to demonstrate the dependence of biological processes on
FGF signaling. In several studies the dominant-negative FGF receptor 1 (XFD,
FGFR1) was used to block FGF receptor 1 (FGFR1)-mediated FGF signaling.
From injections of mRNA encoding
FGFR1 into Xenopus embryos,
it was demonstrated that FGFs are involved in mesodermal induction
(Amaya et al., 1991
;
Amaya et al., 1993
;
Cornell and Kimelman, 1994
;
LaBonne and Whitman, 1994
).
This conclusion was supported by transgenic expression of this
dominant-negative mutant receptor in Xenopus embryos
(Kroll and Amaya, 1996
). The
effects of FGF signaling on neural induction are less clear. FGFs can induce
neural differentiation in dissociated ectodermal cells
(Kengaku and Okamoto, 1993
),
and expression of neural markers in ectodermal explants
(Barnett et al., 1998
;
Launay et al., 1996
;
Sasai et al., 1996
). However,
no significant effects on neural induction were observed when
FGFR1 was
expressed in transgenic Xenopus embryos
(Kroll and Amaya, 1996
). More
recently a dominant-negative FGF receptor 4a (
FGFR4a) has been used to
investigate the role of FGF signaling in embryogenesis. This dominant-negative
receptor is a better inhibitor of FGF signaling than
FGFR1 (XFD) and
inhibition of FGFR4a results in different effects on embryogenesis than does
the inhibition of the FGFR1 mediated pathway
(Hongo et al., 1999
). In
contrast to the overexpression of
FGFR1, the overexpression of
FGFR4a has been shown to impede neural induction in Xenopus
embryos, demonstrating that individual FGF signaling pathways have distinct
roles in the formation of different tissues
(Hardcastle et al., 2000
;
Hongo et al., 1999
). These
studies highlighted the acute need to evaluate each FGF signaling pathway
separately, because pathways mediated by different receptors might have
different effects on embryologic events. In this paper, we analyze the effects
of the elimination of FGF signaling through FGFR4a on the development of
retinal cell types.
During eye development, the initially undifferentiated, seemingly
homogeneous, retinal progenitor cells develop into a layered array of seven
cell types with different capabilities. These include the light sensitive
photoreceptor cells, the bipolar interneurons that transmit electrical
stimulus from the photoreceptor to the ganglion cells, and the ganglion cells
that transmit the information from the eye to the brain. The formation of
these cells types, and their correct proportionality, is necessary for the
proper function of the vertebrate eye. FGF molecules and their receptors are
expressed in developing retina (de Iongh
and McAvoy, 1993; Gao and
Hollyfield, 1995
; McFarlane et
al., 1998
; Patstone et al.,
1993
), and several studies have investigated their role in
proliferation, survival and differentiation of retinal cells
(Guillemot and Cepko, 1992
;
Park and Hollenberg, 1989
;
Sievers et al., 1987
). It was
shown that inhibition of the FGFR1-mediated pathway in Xenopus
embryos resulted in a 50% loss of photoreceptor and amacrine cells, with a
concomitant increase of Müller cells
(McFarlane et al., 1998
). The
role of FGF signaling through receptors FGFR1 and FGFR2 in the survival of
murine photoreceptor cells has also been investigated
(Campochiaro et al., 1996
).
These authors demonstrated that inhibition of FGF signaling using
FGFR1
and
FGFR2 resulted in a progressive death of photoreceptor cells. The
role of FGFR4a-mediated FGF signaling on retinal development has not yet been
examined. In this study, we have used the regulatory sequences of the
Xrx1A gene to direct
FGFR4a expression in retinal progenitor
cells of transgenic Xenopus embryos. This approach has a significant
advantage over the injection of dominant-negative receptor mRNA into
Xenopus embryos, as it does not interfere with neural induction. This
allows us to monitor the role of FGFR4a-mediated signaling on the development
of retinal cells after the retinal progenitor cells were formed. Using
transient transgenic lines of Xenopus laevis, we have found that the
specification of retinal cells, as well as eye development on the whole, is
severely affected in transgenic embryos expressing the
FGFR4a
construct. These embryos have abnormal retinal layering and the population of
photoreceptor cells is either absent or significantly reduced. At the same
time the percentage of Müller glial cells is significantly increased.
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MATERIALS AND METHODS |
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Constructs for transgenesis
GFP reporter plasmids pCS2mt+SGP and pCS2mt+UGP were kindly provided by Dr
Mike Klymkowsky (Rubenstein et al.,
1997). The plasmid phs3LSN, containing a minimal heat shock
protein gene promoter, was a gift from Dr Janet Rossant. A
HindIII-Asp718 fragment containing GFP from pCS2mt+SGP was
subcloned into pBluescriptIIKS to generate pBS-GFP for further transgene
construction. A detailed description of the Xrx1A deletion constructs
is in the legend of Fig. 3.
Fgfr4a was prepared by digestion of Fgfr4a cDNA with
BglII and self ligation, removing the 216 amino acid kinase domain
(Golub et al., 2000
). To
prepare Xrx1A-
Fgfr4a, the
Fgfr4a
coding region was amplified from
Fgfr4a cDNA by PCR (forward:
5'-CCCATGATCACATGTCTGGATCCATAAG-3'; and reverse:
5'-GATCATCGATAAGTCCCAAGTTCACTGTG-3'), digested with BclII
and ClaI, and ligated to the BamHI and ClaI sites
of the GFP construct, pCS2mt-UGP
(Rubenstein et al., 1997
). The
Fgfr4a-GFP fusion was then subcloned into the HindIII
and NotI sites of construct 1 (replacing the GFP cassette) in two
sequential steps (as HindIII-NotI, and HindIII fragments).
DNA was prepared for transgenesis by digestion with SacI, or
SacI and NotI. In Xrx1A regulatory sequence
analysis experiments, negative expression results with transgene constructs 6,
8, 9, 10 and 11 were confirmed by PCR using the GFP-specific primer
5'-GAATTGGGACAACTCCAGTG-3', and the Xrx1A regulatory
element specific primers 5'-GAACGACACAAAGGACACAG-3' (239-220) or
5'-GTACAAAGGTAGAGAAGCAG-3' (666-647).
|
The sequence of the 3.4 kb Sst I-PstI fragment that was used for the deletion analysis of the Xrx1A regulatory sequences can be found at GenBank Accession Number AY250711.
In situ hybridization and immunostaining
Whole-mount in situ hybridization was performed as described by Smith and
Harland (Smith and Harland,
1991). Staining of paraffin sections, frozen sections and whole
embryos with antibodies was performed as previously described
(El-Hodiri et al., 1997
).
Primary and secondary antibodies were used at the following dilutions: mouse
anti-rhodopsin (Adamus et al.,
1991
), 1:100; rabbit anti-calbindin (Swant), 1:400; rabbit
anti-Islet-1, 1:100; mouse anti-glutamine synthetase (Chemicon), 1:100;
HRP-conjugated goat anti-mouse IgG (Sigma), 1:100; Cy3-conjugated goat
anti-mouse IgG (Jackson ImmunoResearch), 1:200; and Cy3-conjugated goat
anti-rabbit IgG (Jackson ImmunoResearch), 1:200. Nuclei were counterstained
with either 5 µg/ml Hoechst 33258 (Sigma) or 2 µM Topro-3 (Molecular
Probes). Stained sections were examined and images recorded using either a
Zeiss 510 LSM confocal microscope, or a Leica MPS52 fluorescent microscope and
Diagnostic Instruments digital camera. For the analysis of the percentage of
specific retinal cell types, the glutamine synthetase-positive and
Islet1-positive cells were counted in transgenic embryos at stage 42 on 12
µm serial sections across the retina.
TUNEL staining
Apoptotic cells were detected by whole-mount TUNEL staining following a
previously described protocol (Hensey and
Gautier, 1997) with some modifications. The embryos were
rehydrated in PBS/0.5% Tween-20 (2x20 minutes), then washed in PBS and
terminal deoxynucleotidyl transferase (TdT) buffer. The labeling reaction was
carried out in TdT buffer containing 0.5 µM digoxigenin-11-dUTP (Roche) and
150 U/ml TdT (Invitrogen). After termination of the reaction and PBS washes,
the embryos were washed in PBS/0.1% TritonX-100/0.2% BSA (PBTxB buffer)
and blocked in PBTxB buffer with 20% goat serum. The embryos were then
incubated with 1:2000 anti-digoxigenin antibody coupled to alkaline
phosphatase (Roche), and then washed in PBTxB buffer (4x30
minutes). The stain was developed in alkaline phosphatase buffer using
nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate substrates.
The stained embryos were dehydrated in ethanol, counterstained with Eosin,
embedded in paraffin wax and 10 µm sections cut. Sections were de-waxed in
xylene and mounted with Permount (Fisher). Stained cells were counted in every
second section across the entire retina.
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RESULTS |
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Although deletion to -1304 bp abolished the early GFP expression that takes place during neurulation, expression of GFP was still present in tadpole eyes, suggesting the presence of a second regulatory region, which controls the late expression of the Rx gene. This late expression initiated around stage 35 and was primarily limited to the photoreceptor cells. We made several deletion constructs to identify the sequences responsible for this late onset of GFP activity. We found that deletion to -982 bp (construct 5) had no effect on expression of GFP in the photoreceptor cells. However, a deletion to -606 bp (construct 6) eliminated all activity of the Xrx1A enhancer, which suggests that the -982 to -606 bp region contained cis-acting elements required for the photoreceptor activity of the Xrx1A promoter.
To test this hypothesis further, we fused the -982 to -606 bp region to a minimal heat shock protein promoter (hsp; construct 7) and produced transgenic animals. This construct was active in photoreceptor cells confirming that the -982 to -606 bp region was sufficient for the activity of the Xrx1A promoter in photoreceptor cells. Comparison of expression data from construct 6 with that from construct 7 also indicated that the region from 0 to -606 bp contained the Xrx1A core promoter sequences. This was confirmed by the finding that the -982 to -606 bp region was not able to direct GFP expression to the photoreceptor cells in the absence of the heat shock protein promoter (construct 8). The heat shock promoter alone was also inactive when placed in front of the GFP coding region (construct 9).
Further deletions in this crucial region resulted in abolishment of GFP expression. For example, a deletion to -857 bp (construct 10) established that the region from -982 to -857 bp is essential for gene activity. However, this segment alone was not sufficient to activate GFP expression. When this short fragment of DNA was placed in front of the hsp promoter (construct 11), it did not activate the Xrx1A promoter. However, the -982 to -606 bp region crucial for the late activation of the Xrx1A promoter is not necessary for the early activation of the Xrx1A promoter. We have deleted the -1304 to -606 bp sequence from construct 1 (to create construct 12) and found that GFP expression was activated during neurulation, as it was with construct 1. This was the case regardless of whether the putative Xrx1A core promoter sequences were used (construct 12) or whether they were replaced by the hsp promoter (construct 13). Interestingly, transgenic animals carrying constructs 12 and 13 displayed GFP fluorescence in their photoreceptor cells, suggesting that the SstI-BamHI fragment contains regulatory elements that can direct or stabilize gene expression in the photoreceptor cells. Therefore it appears that the SstI-PstI fragment contains two independent regulatory sequences that can direct gene expression to the photoreceptor cells.
Effect of FGF signaling on specification of retinal cell types
To investigate the role of FGFR4-mediated signaling in retinal development,
we used the newly characterized Xrx1A regulatory sequences (a 3.4 kb
SstI-PstI fragment from construct 1;
Fig. 3) to drive expression of
a dominant-negative FGF receptor 4a (Fig.
4A) in the developing retina of Xenopus. Initially, a
construct was made that linked the coding region of the dominant-negative
receptor construct in frame to the coding region of GFP. Unfortunately, this
fusion protein failed to fluoresce when exposed to UV light and therefore
could not be used to detect transgenic embryos. Based on the suggestion of
Bronchain et al. (Bronchain et al.,
1999), we inserted a stretch of glycine residues upstream of the
GFP-coding region. However, as this did not alleviate the problem, we
co-injected
Fgfr4a-GFP with a cardiac actin
promoter-GFP (Car-GFP) construct, which drives expression in
the skeletal and heart muscle where Xrx1A is never expressed. This
allowed us to easily identify transgenic embryos. Definitive genotyping of
these embryos was accomplished by PCR. More than 75% of embryos carrying the
Car-GFP construct were also transgenic for the
Fgfr4a-GFP construct.
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This experiment demonstrated that the problem in the FGFR4a
transgenic retinas was with the initial formation of photoreceptor cells,
rather than with their survival. If the expression of
FGFR4a affected
photoreceptor survival, we would have expected to see normal numbers of
photoreceptor cells in early embryonic stages, followed by a reduction in
their numbers. This was clearly not the case. To further eliminate the
possibility that the lack of photoreceptor cells was caused by their selective
cell death, we performed TUNEL assays on stage 29 and stage 35 embryos. As
demonstrated in Fig. 6, the
rates of retinal cell death in stage 29 embryos were practically identical in
single- (58) and double-transgenic animals (54). There was a slight increase
in cell death in double-transgenic animals at stage 35 that was not
statistically significant. These results suggested that the absence of
photoreceptor cells at later stages of development was not caused by increased
death of photoreceptor progenitor cells, but rather by the lack of their
specification.
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DISCUSSION |
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Xrx1A is one of the earliest and most specific markers of retinal
development. It is expressed in the anterior neural plate, in cells that will
become retinal progenitor cells. We have found that the 3.2 kb 5'
upstream DNA sequence of the Xrx1A gene contains regulatory regions
sufficient to direct gene expression in the developing retina. Expression of
GFP directed by these regulatory sequences starts in the anterior neural plate
and persists in the retina, at least until stage 42. Whereas before stage 30
GFP expression was present in all retinal cell types, after stage 30 the GFP
expression became progressively restricted to the photoreceptor cells.
Therefore this region can account for the early transcription of
Xrx1A in the anterior neural plate and retinal progenitor cells
(Casarosa et al., 1997;
Mathers et al., 1997
), and for
the late expression in photoreceptor cells
(Perron et al., 1998
). At
later stages, GFP expression was not completely limited to photoreceptor
cells, as some cells outside of the photoreceptor layer displayed
fluorescence. The identity of these cells is not known at present. One
attractive hypothesis is that these are photoreceptor progenitor cells. As we
do not have an independent specific marker for progenitors of photoreceptor
cells, we cannot currently test this hypothesis.
Deletion analysis of the 3.2 kb segment of upstream sequence revealed that this DNA segment can be divided two distinct regulatory regions. The first region, -2726 to -1304 bp upstream of the PstI site, could activate transcription early in the anterior neural plate, whereas the second region, which is located -982 to -606 bp upstream of the PstI site, could activate transcription late in the development of photoreceptor cells. There appears to be a redundancy in photoreceptor specific regulatory elements, as the first region alone was sufficient for the restriction of GFP expression to the photoreceptor cells (Fig. 3; construct 12 and 13). Therefore both regions can direct gene expression in the photoreceptor cells, but only the first region can direct gene expression in the retinal progenitor cells.
We have taken advantage of these sequences to study the role of
FGFR4a-mediated FGF signaling on the development of retinal cell types. For
this purpose, we expressed the dominant-negative FGFR4a under the control of
the Xrx1A regulatory sequences in retinal progenitor cells of
transgenic Xenopus laevis. Our results showed that FGF signaling
mediated by FGFR4a was essential for normal specification of retinal cell
types and for the correct development of the entire retina. Specifically, we
observed a marked loss of photoreceptor cells in transgenic animals expressing
FGFR4a. This demonstrated that FGFR4a-mediated FGF signaling in the
retina is required for the specification of photoreceptor cells, as measured
by expression of rhodopsin and calbindin. At the same time, we observed an
increase in the formation of Müller glial cells, whereas the number of
glutamine synthetase-positive ganglion/amacrine cells was not significantly
affected. As it was demonstrated that these altered ratios were not due to
selective cell death of specific retinal cell types, we conclude that the lack
of photoreceptor cells was caused by a failure in photoreceptor
specification.
This observation agrees well with other studies that have investigated the
role of different FGF molecules in photoreceptor formation. McFarlane et al.
demonstrated that inhibition of FGF signaling using a dominant-negative form
of XFGFR1 (XFD) resulted in a 50% loss of photoreceptor cells with a
concurrent 3.5-fold increase in Müller glial cells
(McFarlane et al., 1998),
suggesting a shift towards a Müller cell fate in the absence of a
Fibroblast Growth Factor receptor signal. In addition, Hicks and Courtois
showed that FGF can increase the number of photoreceptor cells in dissociated
rat retinal cells (Hicks and Courtois,
1992
). The experiments of Campochiaro et al. showed that
transgenic mice expressing dominant-negative forms of FGFR1 and FGFR2 in
photoreceptor cells displayed progressive photoreceptor degeneration, but did
not show abnormalities in the specification of photoreceptor cells
(Campochiaro et al., 1996
).
This result differs from ours in that the development of photoreceptor cells
was not affected, only their survival. However, this is not surprising as the
rhodopsin promoter used by Campochiaro et al. activates gene expression only
after the photoreceptor cells are differentiated to a significant degree.
Taken together, all published observations on the role of FGF signaling in
retinal development present a unified picture, in which FGF signaling is
necessary for specification and survival of retinal cell types. In addition,
we believe that FGF signaling is necessary for the correct layering of the
retina.
FGF molecules have also been suggested to play a role in the separation of
the neuroepithelium of the optic vesicle into the neuroretina and retinal
pigment epithelium (RPE). Hyer et al. found that in the absence of surface
ectoderm, the neural and RPE cells were mixed together
(Hyer et al., 1998).
Exogenously added FGF1 was able to replace the function of the surface
ectoderm. In a similar study, Nguyen and Arnheiter demonstrated that FGF1- or
FGF2-coated beads could transform retinal pigment epithelium into the
neuroretina (Nguyen and Arnheiter,
2000
). Based on these observations, we would have expected a
conversion of the neuroretina into RPE in our transgenic animals that express
the
FGFR4a in the developing retina. We did not observe such a
conversion. This might be because we have not eliminated FGF signaling
mediated by other FGF receptors. Although this is a formal possibility, the
current belief is that
FGFR4a does not inhibit only one specific FGF
signaling pathway, but can also inhibit all other FGF signaling pathways. This
is because
FGFR4a can heterodimerize with other FGF receptors. If this
is indeed the case, a general inhibition of FGF signaling might result from
such interactions. However, it was shown by Hongo et al. that although
FGFR4a is capable of inhibiting FGF signaling effectively, it is not
able to do it completely (Hongo et al.,
1999
). We may not observe a conversion of neuroretina into RPE
because the primary molecule that leads to a separation of neuroretina and RPE
is not a FGF molecule. The experiments of Hyer et al., and of Nguyen and
Arnheiter, do not exclude this possibility
(Hyer et al., 1998
;
Nguyen and Arnheiter, 2000
).
Indeed, a targeted elimination of FGF1, or FGF2, or both in mice
(Dono et al., 1998
;
Miller et al., 2000
;
Ortega et al., 1998
) did not
result in the retinal phenotype observed by Hyer et al., and by Nguyen and
Arnheiter (Hyer et al., 1998
;
Nguyen and Arnheiter, 2000
),
indicating that further experimentation will be necessary to fully understand
the role of FGF molecules and their receptors in retinal formation.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Current address: Division of Molecular and Human Genetics, Children's
Research Institute, Columbus, OH 43205, USA
Current address: MCB, Harvard University, Cambridge, MA 02138, USA
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