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First published online 5 November 2008
doi: 10.1242/dev.024620
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1 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
2 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
3 Department of Ophthalmology, Harvard Medical School, Boston, MA 02115,
USA.
* Author for correspondence (e-mail: cepko{at}genetics.med.harvard.edu)
Accepted 2 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Alternative splicing, Sfrs1, Survival of retinal neurons, Temporal
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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23,049)
(http://www.ensembl.org/Mus_musculus/index.html)
is similar to that in Arabidopsis thaliana
(Seki et al., 2002
). Thus, it
has been proposed that the complexity of higher metazoans must arise via the
regulation of these genes at the transcriptional and post-transcriptional
level. Alternative splicing (AS) is the mechanism by which exons of a single
gene can be spliced in various combinations to encode a diverse set of
proteins. Indeed, 74% of all human genes are known to be alternatively spliced
and different tissues exhibit varying degrees of AS
(Cheng et al., 2005;
Johnson et al., 2003;
Kampa et al., 2004). Moreover,
when one considers that a single gene can produce multiple isoforms, the
number of proteins encoded by the genome will most likely be much higher than
the current estimates. In humans, the mRNAs that are expressed in the central
nervous system (CNS) are subjected to the highest degree of AS when compared
with other mature tissues (McCullough et
al., 2005). However, the role of AS in CNS development is
relatively unexplored. Given the complexity of AS, combined with that of CNS
development, it is important that a system employed to investigate this
question should be accessible, well characterized during development and
relatively amenable to functional perturbation.
The vertebrate retina is part of the CNS, yet is a relatively simple tissue
with six neuronal cell classes (rod photoreceptors, cone photoreceptors,
horizontal cells, bipolar cells, amacrine cells and ganglion cells) and one
glial type (Müller glia) organized in a stereotypic manner. The birth
order of each cell type is conserved, such that ganglion cells, cone
photoreceptors and horizontal cells are among the first-born cell types,
followed by amacrine cells, rod photoreceptors, bipolar cells and Müller
glia (Rapaport et al., 2004;
Sidman, 1961;
Young, 1985a). The production
of each postmitotic cell type begins in the central retina and expands from
the center to the periphery (Rapaport et
al., 2004; Young,
1985a; Young,
1985b). Moreover, retinal development has been the focus of many
studies, leading to a better understanding of mechanisms that govern cell fate
determination and differentiation (Cepko,
1996; Livesey and Cepko,
2001). Thus, the current investigation focuses on understanding
the role of an alternative-splicing factor (ASF) called splicing factor
arginine/serine-rich 1 (Sfrs1) in retinal development.
Sfrs1 belongs to a highly conserved arginine/serine-rich (SR)
protein family of RNA processing factors found throughout metazoans and in
plants (Zahler, 1999;
Zahler et al., 1992). The role
of Sfrs1 in splicing has been well documented, but only recently has
its role in any developmental context been investigated
(Xu et al., 2005). In C.
elegans, ablation of the Sfrs1 homolog resulted in late
embryonic lethality, suggesting that its function is non-redundant in at least
one critical stage of development (Kawano
et al., 2000; Longman et al.,
2001). In mice, the loss of Sfrs1 also resulted in
embryonic lethality (Xu et al.,
2005). A conditional knockout (cKO) mouse in which the
Sfrs1 gene was flanked with loxP sites
(Sfrs1fl/fl) has enabled functional studies of
Sfrs1. Xu et al. have shown that loss of Sfrs1 does not
cause aberrant proliferation and/or cell death of cardiac progenitor cells and
that embryonic heart development proceeds normally
(Xu et al., 2005). However,
during postnatal remodeling of the heart, aberrant splicing of specific target
genes such as cardiac troponin T (cTnT; Tnnt2 - Mouse Genome
Informatics), the Z-line protein cypher (Ldb3) and
Ca2+/calmodulin-dependent kinase II 
(CaMKII; Camk2d), resulted in physiological defects
in the heart (Xu et al.,
2005).
In this report, we show that Sfrs1 is expressed in the developing
mouse retina and is itself regulated by AS. We have identified a new isoform
that is expressed during late embryonic development and continues to be
expressed during postnatal retinal development. This novel isoform lacks the
SR domain that is crucial for the nuclear localization of the Sfrs1 protein
(Kataoka et al., 1999;
Lai et al., 2000;
Lai et al., 2001).
Furthermore, the present investigation employs the
Sfrs1fl/fl mice created by Xu et al. to determine the role
of Sfrs1 in the developing mouse retina. To specifically ablate
Sfrs1 function during retinal development, the
Sfrs1fl/fl mice were crossed to mice that express Cre
recombinase under the regulation of the ceh-10 homeodomain-containing homolog
(Chx10; Vsx2), a gene that is expressed in retinal
progenitor cells (Rowan and Cepko,
2004). The loss of Sfrs1 function resulted in a small eye
at birth. We found that loss of Sfrs1 function did not have a
significant effect on proliferation. However, neurons generated during early
embryonic development underwent apoptosis, whereas those generated after birth
did not. Consequently, neurons generated in the embryo, such as ganglion
cells, cone photoreceptors, horizontal cells and amacrine cells, were
significantly reduced in the Sfrs1-cKO retina. By contrast, rod
photoreceptors, bipolar cells, late-born amacrine cells and Müller glia
survived in the Sfrs1-cKO retina. The subset of susceptible neurons
was defined primarily by the time of their birth, a possible reflection of the
temporal heterogeneity in gene expression that has been defined for retinal
progenitor cells (Trimarchi et al.,
2008).
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). The presence of the Cre allele was also confirmed
by PCR (Rivera-Feliciano and Tabin,
2006). When mice were subjected to surgical procedures, all IACUC
protocols to minimize pain during and after surgery were observed.
RT-PCR
Retinae from different developmental time points were harvested and total
RNA prepared in Trizol following the manufacturer's protocol (Invitrogen). For
cDNA synthesis, 5 µg of total RNA from retinae harvested at various time
points was used (Kanadia et al.,
2006). PCR to detect Sfrs1 isoforms was performed with a
forward (5'-ATGTCGGGAGGTGGTGTGATCC-3') and a reverse
(5'-CCAATCATCTTATGTACGAGAGCGAGATC-3') primer for 30 cycles
(95°C for 35 seconds; 58°C for 25 seconds; 68°C for 2 minutes).
PCR to detect Tnnt2 isoforms was performed as described previously
(Kanadia et al., 2003).
In situ hybridization on sections and dissociated cells
In situ hybridization (ISH) on 16 µm cryosections and dissociated cells
was performed as described previously
(Trimarchi et al., 2007). All
the probes used in this report are as published previously
(Trimarchi et al., 2008; Trimarchi et al.,
2007). For Sfrs1 we employed a 3'UTR probe
corresponding to bp 1620-2639 in the clone NM_173374 in the NCBI database.
Immunofluorescence
For immunofluorescence (IF), 16 µm cryosections were first hydrated in
phosphate-buffered saline (PBS, pH 7.4), followed by the immunohistochemistry
protocol of Kim et al. (Kim et al.,
2008). The dilutions of the primary antibodies were: chicken
anti-GFP (1:2000) (Abcam); mouse anti-Pax6 (1:300) (Covance); rabbit
anti-Chx10 (1:300) (Cepko laboratory); mouse anti-rhodopsin (4D2; 1:300)
(Molday and MacKenzie, 1983);
mouse anti-glutamine synthetase (1:300) (Chemicon); rabbit anti-red/green
opsin (1:300) (Chemicon); and mouse anti-Ki67 (1:250) (BD Pharmingen).
Immunoblot
The nuclear/cytoplasmic extraction protocol from Pierce was employed on
retinae (n=10) from different stages. Upon fractionation, 30 µg of
protein was resolved on a 4-20% Tris-glycine gradient gel (Invitrogen),
followed by transfer of the proteins to a positively charged nylon membrane
(Invitrogen), which was then subjected to immunoblot analysis as described
previously (Kanadia et al.,
2006). The primary antibodies used were mouse anti-Sfrs1 (1:1000)
(Lifespan) and mouse anti-Cugbp1 (1:500) (Abcam).
Electron microscopy
P0 pups were harvested in 0.1 M sodium cacodylate buffer (pH 7.4), followed
by fixation in 2% paraformaldehyde (PFA) and 2.5% glutaraldehyde in 0.1 M
sodium cacodylate buffer. Retinae were then processed by the Harvard Medical
School Electron Microscopy Core Facility.
P0 electroporation
P0 pups were electroporated with pCAG-Cre and pCAG-LoxP-Stop-LoxP-GFP
plasmids or pCAG-GFP plasmid alone
(Matsuda and Cepko, 2004).
Viral infections
Retroviral vectors were generated by cloning Cre after the IRES in
the pQC-H2B-GFP-IRES-MCS vector (Punzo and
Cepko, 2008). All viruses were prepared as described previously
(Cepko, 1989). E10 viral
infections by ultrasound-assisted delivery in timed-pregnant
Sfrs1fl/fl and/or Sfrs1wt/wt females
were performed as described previously
(Punzo and Cepko, 2008). P0
viral infections were performed as described previously
(Matsuda and Cepko, 2004).
BrdU pulse labeling
A pregnant female at E16 was weighed and then injected with 3 µg of
BrdU/g body weight. At P7, retinae were harvested and processed for BrdU
staining by antigen retrieval as described by the manufacturer (Vector Labs).
Next, the slides were fixed in 4% PFA for 20 minutes followed by two 5-minute
washes with PBS. Slides were treated with 2M HCl for 30 minutes at room
temperature, followed by a brief wash with 0.1 M boric acid (pH 8.5) and IF
was performed as described previously (Kim
et al., 2008).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In addition, the ISH signal for Sfrs1 was enriched
in cells at the apical end of the retina
(Fig. 1A, E10.5 arrowhead),
where the retinal progenitor cells undergo the M phase of their cell cycle
(Sidman, 1961;
Young, 1985b). Similar
asymmetric enrichment of the ISH signal for Sfrs1 was observed in the
olfactory epithelium (see Fig. S1 in the supplementary material). ISH for cell
division cycle 20 (Cdc20), a marker of the G2-M phase of the cell
cycle, and IF for phosphorylated histone H3 (PH3), a marker of M phase, were
employed (see Fig. S1 in the supplementary material) to determine whether the
region of Sfrs1 expression in the olfactory epithelium was also the
region of M phase (Kawauchi et al.,
2005; Trimarchi et al.,
2008; Weinstein,
1997). As predicted, both the ISH for Cdc20 and IF for
PH3 confirmed that the mRNA for Sfrs1 was enriched in areas
expressing these genes, thereby suggesting the involvement of Sfrs1
in cells in the G2-M phase of the cell cycle. At E12.5, Sfrs1
expression was expanded to the entire neuroblastic layer (NBL)
(Fig. 1A) and was also observed
in the lens epithelium. In addition, at E13.5, expression of Sfrs1
was transiently enriched in cells lining the vitreal side of the peripheral
retina (Fig. 1A, E13.5,
arrowhead), but this expression pattern was no longer observed by E14.5, at
which time the expression of Sfrs1 in the ONBL persisted. At P0, the
expression of Sfrs1 was again in the ONBL
(Fig. 1A). During postnatal
development, the expression of Sfrs1 was restricted to the newly
forming inner nuclear layer (INL) in the central retina, while in the
periphery the expression of Sfrs1 was observed in progenitor cells.
At P6, Sfrs1 expression became restricted to the lower half of the
INL, where mostly amacrine and displaced ganglion cells are found, and to the
ganglion cell layer (GCL), where ganglion and displaced amacrine cells are
found (Fig. 1A). This pattern
was also observed at P10 and P30 (Fig.
1A).
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; Ni et al.,
2007). Thus, we investigated whether AS might modulate the protein
levels of Sfrs1 during retinal development. To determine the AS status of
Sfrs1, which has 5 exons (Fig.
1B), a forward primer in exon 1 (start codon) and reverse primer
in exon 4 (stop codon) (Fig.
1B, red arrows) were utilized. Indeed, Sfrs1 was found to
be regulated via AS in a temporal manner, such that only one isoform (Sfrs1b)
was observed in the embryo, whereas an additional isoform at a higher
molecular weight (Sfrs1a) was also expressed postnatally
(Fig. 1C). The temporal
regulation of Sfrs1 via AS led us to investigate whether a similar
form of regulation was also employed during the development of other tissues.
Because the role of Sfrs1 in heart development has been investigated
(Xu et al., 2005), this tissue
was examined. RT-PCR analysis with the same primers showed that during
embryonic heart development, both isoforms of Sfrs1 were present. By
contrast, the predominant isoform at P2 was the Sfrs1b isoform, which was also
the only one expressed in the adult (Fig.
1D).
Sequence analysis revealed that Sfrs1b was the canonical isoform, whereas
Sfrs1a retained intron 3. Consequently, in the Sfrs1a isoform there was a
frame shift in the coding sequence resulting in the truncation of the RS
domain. This change is of functional significance because Sfrs1 shuttles
between the cytoplasm and the nucleus and the phosphorylation of the RS domain
is required for its nuclear localization
(Caceres et al., 1998;
Ma et al., 2008). Thus, during
retinal development, the AS of Sfrs1 produces an isoform that would
most likely fail to translocate into the nucleus. In agreement with the RT-PCR
analysis, immunoblot analysis showed two immunoreactive bands in the
cytoplasmic fraction from the postnatal, but not the embryonic, retina
(Fig. 1E). In addition, the
lower molecular weight isoform (Sfrs1a) of Sfrs1 was significantly
more abundant in the cytoplasmic than in the nuclear fraction
(Fig. 1E). Since the Sfrs1a
isoform was predicted to be cytoplasmic, the low-level detection of Sfrs1a in
the nuclear fraction might be due to cross-contamination between the two
fractions. The inability of the Sfrs1a isoform to shuttle into the nucleus was
further investigated by fusing the coding sequence of Sfrs1a and Sfrs1b to
GFP, followed by transfection of each plasmid into NIH3T3 cells. As predicted,
the canonical isoform, Sfrs1b, was predominantly located in the nucleus,
whereas Sfrs1a was not observed in the nucleus
(Fig. 1F). In summary,
Sfrs1 is expressed throughout retinal development and is regulated by
AS in a temporal manner.
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).
Consequently, the majority of the developing retina lacked Sfrs1
function. The Sfrs1-cKO retinae underwent aberrant embryonic
development, which was inferred from the observation that Sfrs1-cKO
mice were born with small eyes (Fig.
2A). Sfrs1-cKO eyes also had a thin optic nerve and the
total eye weight was half that of the wild type
(Fig. 2B,C). Hematoxylin and
Eosin (H&E) staining of the retinal sections revealed a significant
decrease in the size of the retina, but not in the retinal pigmented
epithelium (RPE) or the lens (Fig.
2D,E). The retina was detached from the RPE
(Fig. 2E, arrowhead) while
abutting the lens and exhibited a marked decrease in the cellularity of the
GCL, along with a perturbed inner limiting membrane (ILM)
(Fig. 2G, arrowhead).
Quantification of the number of nuclei in the GCL
(Fig. 2F,G, arrowhead)
confirmed the decrease in GCL cellularity
(Fig. 2H), which in turn was
reflected in the thin optic nerve (Fig.
2B, insets). Ultrastructural analysis showed that cells in the
mutant retina were highly disorganized, with vacuolar inclusions, unlike the
wild-type retina, which had elongated nuclei with a defined polarity
(Fig. 2I-L). The absence of the
ILM in the H&E-stained sections was confirmed by ultrastructural analysis
(Fig. 2L, arrowhead). The
mutant retina exhibited a dysmorphic fibrous layer instead of the
well-organized ILM seen in the wild-type control retina
(Fig. 2K). In summary, loss of
Sfrs1 function had a profound effect on the embryonic development of
the retina and, possibly, also on that of the ciliary body, which together
might have contributed to the microphthalmia observed at birth.
Sfrs1 loss-of-function causes cell death during embryonic retinal development
To determine the cause of the small retina in the Sfrs1-cKO mice
at P0, we investigated whether the mutant retina had a proliferation defect
during embryonic development. Ki67 antibody, a well-characterized
proliferation marker (Gerdes et al.,
1991), was employed on retinal sections from E12.5, E13.5, E18.0
and P0. No proliferation defect was observed in the Sfrs1-cKO retina
(Fig. 3A-H). This was confirmed
by quantification of the PH3 staining in the Sfrs1-cKO retina at
E12.5 (see Fig. S2 in the supplementary material). Therefore, cell death was
investigated as a cause of the small eye phenotype in Sfrs1-cKO mice.
Terminal dUTP nick end labeling (TUNEL) assay revealed a significant increase
in the number of TUNEL+ cells at E12.5, E13.5 and E18.0 in the
mutant retina (Fig. 3I-N).
TUNEL+ cells observed in Sfrs1-cKO retina at E12.5 and
E13.5 were predominantly in the central retina
(Fig. 3J,L), whereas the
majority of the TUNEL+ cells at E18.0 were in the periphery
(Fig. 3N). At P0, few
TUNEL+ cells were observed in the periphery
(Fig. 3P). Furthermore,
quantification of the TUNEL+ cells in the mutant retina at each
stage revealed that the cell death peaked at E13.5, followed by a
decrease in the number of TUNEL+ cells at E18, with few
TUNEL+ cells at P0 (Fig.
3Q-T). In summary, loss of Sfrs1 function during
embryonic development caused cell death and did not cause an observable
reduction in proliferation; however, a subtle reduction in proliferation could
not be ruled out.
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This conclusion was further bolstered by the genetic ablation strategy that
employed the Chx10::Cre line along with a reporter line,
RC::PFWE, in the Sfrs1fl/fl background.
The RC::PFWE strain reports Cre activity by expressing
nuclear β-galactosidase (nlacZ)
(Farago et al., 2006). Thus,
every progenitor cell that expresses Cre should be positive for nlacZ
and negative for Sfrs1 function. The results showed that at least
some retinal progenitor cells did not die, as there were
nlacZ+ neurons in the P7 mutant retina
(Fig. 4E). These neurons, as
judged from their position, were mostly photoreceptors, bipolars and
Müller glia (Fig. 4E). By
contrast, the wild-type littermate retina showed many cells expressing
nlacZ in the ONL (outer nuclear layer) (rod photoreceptors, cone
receptors), INL (bipolar cells, horizontal cells, amacrine cells and
Müller Glia) and in the GCL (ganglion and amacrine cells)
(Fig. 4D). In both the
wild-type and the mutant retinae there were cells that were negative for
nlacZ, which was likely to result from the mosaic expression of
Chx10::Cre (Rowan and
Cepko, 2004). In summary, loss of Sfrs1 function did not
lead to the death of all progenitor cells, although some progenitor cell death
could not be ruled out.
Postmitotic cells are generated in the Sfrs1-cKO retina during early embryonic development
To examine whether the postmitotic cells were dying in the
Sfrs1-cKO retinae, we first determined whether postmitotic cells were
produced. For this, RNA ISH with probes that label differentiated neurons was
employed on E13.5 retinal sections. First, loss of the Sfrs1
transcript in the Sfrs1-cKO retina was confirmed
(Fig. 5B). Although the
majority of the retina lacked Sfrs1, a few cells
(Fig. 5B, arrowhead) were
positive for Sfrs1, most likely reflecting the
Chx10::Cre mosaicism
(Rowan and Cepko, 2004). ISH
analysis was performed on serial sections with probes that mark postmitotic
cells, including neurofilament-like light chain 68 (Nf68;
Nefl - Mouse Genome Informatics), brain 3b (Brn3b;
Pou4f2) and thyroid hormone receptor beta 2
(Trβ2; Thrb). Ganglion cells were produced in
the mutant retina, as shown by the strong ISH signal for Nf68 and
Brn3b (Fig. 5D,F).
Similarly, cones were produced, as shown by the ISH signal for
Trβ2 in the Sfrs1-cKO retina
(Fig. 5H). However,
Trβ2+ cone photoreceptors in the mutant
retina were located throughout the ONBL
(Fig. 5J, arrowhead), rather
than abutting the RPE as was seen in the wild type
(Fig. 5I). In summary, these
data suggest that at least some neurons can be produced and initiate
differentiation in the Sfrs1-cKO retina, with cones showing aberrant
localization.
|
). As
shown in Fig. 6, by P7 there
was a significant reduction in the number of embryonically generated neurons
in the Sfrs1-cKO retina, which suggests that the cell death observed
during embryonic development was most likely that of the postmitotic
cells.
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Sfrs1-cKO retinae undergo further degeneration during postnatal development
The neuronal composition of wild-type and Sfrs1-cKO retinae was
compared at P14, a time when the production of retinal cell types is complete.
Analysis at this stage revealed that the Sfrs1-cKO retina had
undergone further degeneration and exhibited rosettes
(Fig. 8D, arrowhead).
Furthermore, in the mutant, the ciliary body was fused to the retina, such
that it could not be separated from the neural retina
(Fig. 8J, arrowhead), in
contrast to the situation in the wild-type littermate where it was easily
removed (Fig. 8I, arrowhead).
Despite the severe morphological deterioration of the mutant retina at P14, it
still appeared to have a relatively unaltered percentage of Müller glia
as shown by IF with glutamine synthetase antibody
(Fig. 8A,B). Similarly, rod
photoreceptors also appeared relatively unaltered in the mutant retina
(Fig. 8F, upper arrowhead; see
Fig. S3 in the supplementary material). In contrast to the IF signals for
Müller glia and rods, IF for Pax6
(Fig. 8F, lower arrowhead),
Chx10 and red/green opsin showed a significant reduction in the
Sfrs1-cKO retina (Fig.
8C,H). The reduction in the bipolar cells could be secondary to
the loss of cone photoreceptors. This dysmorphic retina ultimately underwent
complete degeneration by P30, as seen by the absence of the entire eye in the
Sfrs1-cKO mouse (Fig.
8L). In summary, the data gathered at P14 indicate that despite
the severe morphological defects observed in the mutant retina, cell types
including rod photoreceptors, Müller glia and bipolar cells were
relatively unaffected, whereas others, such as ganglion cells, amacrine cells,
cone photoreceptors and horizontal cells, seemed to have undergone further
loss.
Sfrs1 is not required for the maintenance of late-born neurons
P0 in vivo electroporation was used to investigate whether rod
photoreceptors, Müller glia and bipolar cells were resistant to the loss
of Sfrs1 function. This strategy used two plasmids, one expressing
Cre and the other expressing GFP in a conditional manner, such that it
reported Cre activity. As shown in Fig.
8, production of rod photoreceptors was normal, and the rod
photoreceptors had proper outer segments
(Fig. 8M). In addition,
amacrine cells appeared to be produced normally. Although bipolar cells and
Müller glia were also produced during this time, the promoter driving GFP
failed to express reproducibly in these two cell types, making it difficult to
assess their relative levels. To investigate the production and maintenance of
bipolar cells and Müller glia, the same virus used for embryonic ablation
of Sfrs1 was used to infect P0 retinal progenitor cells, followed by
analysis at P55. Several nuclear GFP+ clones were observed in the
Sfrs1-cKO retina (Fig.
8O-Q). Based on the position and shape of the nuclei, the
GFP+ cells were judged to be rod photoreceptors, bipolar cells and
Müller glia (Fig. 8O-Q;
see Fig. S4 in the supplementary material). In summary, Sfrs1
function is required in a temporal manner, such that neurons born postnatally
are resistant to the loss of its function.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Sfrs1, an ASF, is not only regulated at the transcriptional level during retinal development, but is also alternatively spliced in a temporal manner (Fig. 1). The newly identified isoform of Sfrs1 includes intron 3, which produces a truncated isoform of Sfrs1 that would fail to translocate into the nucleus. This truncated isoform is observed by immunoblot analysis and the GFP fusion version of this isoform is seen only in the cytoplasm. However, we cannot rule out the possibility that the new isoform is subject to nonsense-mediated mRNA decay and so the true identity of the lower molecular weight isoform warrants further investigation (see Fig. S5 in the supplementary material).
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). It is interesting to note that the presence of the
phenotype in the two tissues is correlated with the absence of the Sfrs1a
isoform. In other words, the times at which the Sfrs1a isoform is expressed
normally are the times when loss of Sfrs1 function does not result in a
phenotype. One possibility is that another ASF that regulates the AS of
Sfrs1 normally compensates for the loss of Sfrs1 function.
Thus, the presence of Sfrs1a serves as a proxy for the presence of the
compensatory ASF, which in the heart is present in the embryo, whereas in the
retina it is present postnatally.
Sfrs1 loss-of-function results in the death of postmitotic cells
Progenitor cells in the Sfrs1-cKO retina did not die in
significant numbers, as proliferation remained unaltered in the
Sfrs1-cKO retina (Fig.
3A-H). In addition, infection of Sfrs1fl/fl
progenitor cells with retrovirus expressing Cre and nuclear-GFP produced
clones consisting mainly of later-born neurons
(Fig. 4C). Similarly, several
nlacZ+ neurons were detected in the P7 mutant retina
(Fig. 4E). The presence of
nlacZ+ cells reflects a history of Cre activity in the
progenitor cells. These data indicate that the loss of Sfrs1 function
does not cause the death of the majority of retinal progenitor cells.
If the loss of Sfrs1 function does not lead to the death of
progenitor cells, then it must result in the death of postmitotic cells. This
possibility is consistent with the pattern of TUNEL+ cells in the
Sfrs1-cKO retinae. Specifically, the pattern of cell death overlaps
with the production of postmitotic cells during development
(Fig. 3)
(Farah and Easter, 2005;
Young, 1985a). Furthermore,
ISH and IF analysis to detect differentiated neurons at P0 and P7 revealed
that ganglion cells, horizontal cells, cone photoreceptors and amacrine cells
were significantly reduced (Fig.
5). Interestingly, these neurons are produced in the embryo,
coincident with the period of greatest cell death in the Sfrs1-cKO
retina. By contrast, the later-born rod photoreceptors, bipolar cells and
Müller glia were not as severely reduced (Figs
5 and
6). Taken together, these data
indicate that in the Sfrs1-cKO retina, the postmitotic cells die in
the embryo, which in turn could affect proliferation, as previous reports
indicate that ganglion cell death can indirectly affect proliferation
(Mu et al., 2005). However, in
the case of the Sfrs1-cKO retina, which had reduced numbers of
ganglion cells, there was little effect on proliferation
(Fig. 5D). It is possible that
the reduced numbers of ganglion cells were sufficient to produce the signals
that might be crucial for proliferation.
The peak of amacrine cell production is E16.5-17.5, and production
ends at P4 (Farah and Easter,
2005; Young,
1985a; Young,
1985b). Given that postnatally generated neurons are not affected
in the Sfrs1-cKO retina, postnatally generated amacrine cells were
not expected to die, which was reflected in the modest reduction in the Pax6
IF signal. By contrast, another marker of amacrine cells, Ndrg4, was
significantly reduced in the mutant retina, which suggests that it marks the
types of amacrine cells produced in the embryo. Based on this interpretation,
we posit that there are at least two classes of amacrine cells as defined by
their birthdate and subsequent dependence on Sfrs1, which warrants
further investigation.
Unlike the aforementioned cell types, rod photoreceptors are born in
significant numbers throughout retinal development. As discussed above, many
of the early-born cells died prior to P0, which was also true for rod
photoreceptors. As shown in Fig.
7, the number of BrdU+ cells in the ONL of the P7
Sfrs1-cKO retina was significantly reduced, suggesting that rod
photoreceptors must have died. The remaining rod photoreceptors could be a
consequence of Chx10::Cre mosaicism. It is unlikely that
this reduction was due to the death of cone photoreceptors because very few
(<5%) are produced at E16 (Carter-Dawson
and LaVail, 1979). By contrast, rod photoreceptor production is
initiated at E13 and by E16 more than 5% of the rod photoreceptors have
been produced. Thus, like amacrine cells, rod photoreceptors produced in the
embryo are most likely to die in the Sfrs1-cKO retina.
Temporal requirement of Sfrs1 function for the survival of retinal neurons
In summary, the current investigation suggests that the key determinant of
whether cells survive in the Sfrs1-cKO retina is when they are born,
as opposed to the subtype of neuron. Several lines of evidence point to this
conclusion. First, the number of TUNEL+ cells decreased late in
embryonic development. Second, the genetic ablation of Sfrs1 early in
development led to the loss of many early-born neurons. This includes the
early-born, but not the late-born, amacrine cells and rods. Third, survival of
postnatal neurons was demonstrated by P0 electroporation of Cre and by P0
viral infection. Survival was coincident with the presence of the Sfrs1b
isoform. Finally, we propose a model in which AS mediated by Sfrs1 is
required for the terminal differentiation and/or maintenance of neurons
produced in the embryo, but not for neurons produced postnatally
(Fig. 9). The data presented
here underscore the dynamic role of ASFs during vertebrate neuronal
development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/23/3923/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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