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First published online 12 November 2008
doi: 10.1242/dev.028308
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1 Sackler Faculty of Medicine, Human Molecular Genetics and Biochemistry, Tel
Aviv University, Ramat Aviv 69978, Tel Aviv, Israel.
2 European Neuroscience Institute, Developmental Neurobiology Laboratory,
University of Göttingen Medical School/Max Planck Society,
Grisebachstrasse 5, 37077 Göttingen, Germany.
3 Albert Einstein College of Medicine, Departments of Ophthalmology and Visual
Sciences and Genetics, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Author for correspondence (e-mail:
ruthash{at}post.tau.ac.il)
Accepted 8 October 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Pax6, Retinal progenitor cells, Retinogenesis, Crx, Cre/loxP
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cell
birth-dating studies have revealed an intriguing sequential program of retinal
cell-type specification, which is highly conserved among vertebrate species.
Invariably, the first cell type to appear in all vertebrates is the ganglion
cell, followed by partial overlap with the appearance of cone photoreceptors,
amacrine and horizontal cells, while the bipolar interneurons and Müller
glia cells appear last. Rod photoreceptor genesis occurs in parallel with that
of the other cell types and peaks around birth
(Carter-Dawson and LaVail,
1979; Rapaport et al.,
2004; Sidman,
1961; Young,
1985).
Based on pivotal cell lineage studies, RPCs were concluded to be inherently
multipotent (Fekete et al.,
1994; Holt et al.,
1988; Turner and Cepko,
1987; Turner et al.,
1990; Wetts and Fraser,
1988). The importance of intrinsic determinants for cell-fate
choices in the retina has been established by cell-dissociation and
heterochronic aggregation experiments.
(Belecky-Adams et al., 1996;
Morrow et al., 1998;
Rapaport et al., 2001;
Reh and Kljavin, 1989;
Watanabe and Raff, 1990).
Based on these findings, the idea has emerged that overall retinogenesis
progresses through gradual shifts in the competence of RPCs to respond to
extrinsic cues (Cepko et al.,
1996). However, the molecular mechanisms that underlie the
postulated competence states of the RPCs remain largely elusive
(Pearson and Doe, 2004).
An important class of cell fate determinants is the family of basic
helix-loop-helix (bHLH) transcription factors, related to the
Drosophila proneural genes atonal and achete-scute
(Brown et al., 2001;
Vetter and Brown, 2001). These
factors were shown to bias progenitor cells toward distinct cell fates
(Inoue et al., 2002;
Wang et al., 2001). A number
of homeodomain transcription factors act in direct conjunction with bHLH
proteins to differentially affect cell-fate choices in RPCs
(Inoue et al., 2002). These
factors are expressed in the proliferating RPCs in conjunction with, and often
preceding expression of the proneural bHLH factors
(Hatakeyama and Kageyama,
2004; Hatakeyama et al.,
2001).
The paired and homeodomain transcription factor Pax6 is a key player in
early eye development across animal phyla
(Halder et al., 1995). This
protein has been shown to control retinal development and cell-fate choices,
which are attributed in part to its regulation of bHLH genes
(Marquardt et al., 2001;
Philips et al., 2005). The
function of Pax6 in mammalian retinogenesis is context dependent. In Pax6-null
embryos, OVs are formed but the subsequent OC morphogenesis is prevented.
Nevertheless, the Pax6-deficient OVs maintain expression of some retinogenic
genes (Rx, Chx10) and appear to undergo premature neurogenesis based
on the expression of pan-neuronal markers
(Baumer et al., 2003;
Grindley et al., 1995;
Marquardt et al., 2001;
Philips et al., 2005). This
premature differentiation, however, is aborted, as fully differentiated
neurons are not identified in the Pax6-deficient optic rudiment
(Philips et al., 2005). In
contrast to the arrested differentiation observed in the Pax6-null mutants,
inactivation of Pax6 at the OC stage results in the exclusive generation of
amacrine interneurons at the expense of all other retinal cell types. Thus, at
this later stage, Pax6 seems to be dispensable for the completion of
neurogenesis but essential for RPC multipotency. The dynamics of amacrine cell
genesis following Pax6 loss from RPCs has never been investigated and thus
additional roles for Pax6 during earlier aspects of retinal cell-fate
specification remain possible.
In this study, we performed a detailed investigation of RPC fate in different genetic Pax6-deficient models in mouse. Our results suggest an early co-existence of two distinct RPC populations that differ in their responsiveness towards Pax6 depletion. These findings therefore suggest a dual requirement for Pax6 in retinal neurogenesis, while uncovering early diversification of RPCs into intrinsically distinct progenitor pools.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The Pax6flox allele contains loxPs flanking the
regions deleted in the Pax6lacZ allele
(Ashery-Padan et al., 2000).
The deletion of the Pax6flox allele by Cre results in the
Pax6del allele (see Fig. S1 in the supplementary
material). The 
-Cre-transgenic line contains the Pax6 P0
promoter and the peripheral retina enhancer (termed ) followed by
Cre which was cloned 5' of IRES-intron-gfp-pA
(Marquardt et al., 2001). The
Chx10-Cre mouse line contains a random integration of the
BAC-Chx10-Cre transgene. This transgene includes a fusion gene of
Cre and GFP, and an internal ribosome entry site (IRES)
followed by human placental alkaline phosphatase (AP). This
GFP-Cre-IRES-AP cassette was inserted into the first exon of
Chx-10 BAC (Rowan and Cepko,
2004). The Z/AP-transgenic mice express the human AP gene
following Cre-mediated excision (Lobe et
al., 1999).
Immunofluorescence and BrdU-incorporation analysis
Immunofluorescence analysis was performed as previously described
(Ashery-Padan et al., 2000).
The primary antibodies were: mouse anti-BrdU (1:100, Chemicon), rabbit
anti-cleaved caspase 3 (1:300, Cell Signaling), goat anti hAP (1:100,
Santa-Cruz), mouse anti Isl1 (1:100, hybridoma-bank), rabbit anti-Pax6
(1:1000, Chemicon), mouse anti-syntaxin (1:500, Sigma) and rabbit anti-VC1.1
(1:500, Sigma). Secondary antibodies conjugated to rhodamine red-X or Cy2
(Jackson Laboratories). BrdU was injected 1.5 hours prior to sacrifice and
conducted as described (Yaron et al.,
2006). Slides were viewed with an Olympus BX61 fluorescent
microscope or laser-scanning confocal microscope CLSM 410 (Zeiss) The image
analysis was conducted with `AnalySIS'.
In situ hybridization
In situ hybridization was performed as previously described
(Yaron et al., 2006). For the
fluorescent in situ hybridization, we employed the HRP-conjugated sheep
anti-digoxigenin Fab fragments (Roche) and the TSA kit (Perkins Elmer).
Measurements of the areas of Crx expression and quantification of BrdU incorporation
To define the borders of the Pax6-Crx+
(region 1) and Pax6-Crx- (region 2), and to
determine BrdU incorporation in each region, three serial sections (10 µm
each) from each eye were analyzed and compared (an example in
Fig. 2). On the first section,
the Pax6 and VC1.1 expression domain was determined using specific antibodies
and on the adjacent section, Crx transcripts were identified using in situ
hybridization. In the Pax6flox/flox;-Cre
mutants, the region which was Pax6-Crx+ was
termed region 1, while the region that was
Pax6-Crx- was termed region 2. On the third
sequential section, the proportion of BrdU+ cells in each region
was determined. This analysis was conducted on three to four eyes for all
genotypes and developmental stages, and for each eye the average value was
calculated from two to four sections (number of eyes indicated in figure
legends). To obtain total cell number in each domain, the measured
4',6-diamidino-2-phenylindole (DAPI; 100ng/ml) area was divided by the
average nucleus size to obtain an estimation of cell number (which was
averaged to be 35 µm2 by measuring the nuclear area for 40
clearly visible cells). The ratio of BrdU+ or caspase 3+
cells from total cell number was calculated for each section. To obtain
control values, we calculated the parameters in the peripheral area of the OC
corresponding to 30% of the length of the outer margin of the OC from the most
distal tip to the optic nerve.
Quantification of the spatial distribution of Crx+Pax6- cells in the Pax6-deficient RPCs of the Pax6flox/flox;Chx10-Cre embryos
Frozen sections were double labeled to detect the expression of Crx and
Pax6 by fluorescent in situ hybridization and immunofluorescence analysis,
respectively. For image analysis, the OC was arbitrarily divided into thirds
based on the length of the outer margin of the OC. The area of
Crx+Pax6- out of the total Pax6- area was
determined in each third (Fig.
5J). This analysis was conducted on central sections from six
Pax6flox/flox;Chx10-Cre eyes (11 sections in total).
Chromatin immunoprecipitation (ChIP)
Isolated mouse embryo eyes or limbs (E13) were used as a tissue source for
ChIP. The dissociated cells were crosslinked in 1% formaldehyde for 15 minutes
at room temperature. The ChIP-PCR was performed on 
100 eyes or 40 limbs
according to the manufacturer's protocol (Upstate Biotechnology). The
immunoprecipitations were performed overnight at 4°C using 5 µg of
rabbit anti-Pax6 polyclonal IgG (Covance) or 5 µg of normal rabbit IgG
(Santa Cruz Biotechnology). The PCR primer pairs used for the ChIP assay were:
for the detection of Crx promoter, 5'TAAGCAGACGGTGCCCTTCC-3'
(forward), 5'-AGGAAATAGGTCCCCTCACAC-3' (reverse); and for the
detection of the Crx 3' UTR untranslated region,
5'-CACACCAGGAAAGGGCATGG-3' (forward),
5'-TCTGCCTCTACCTCCCTCGTG-3' (reverse).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Here,
we performed a detailed investigation of Pax6-deficient OV retinal
precursors, focusing on a potential regulatory relationship between Pax6
activity and the pathways for photoreceptor and amacrine specification
(Garelli et al., 2006;
Marquardt et al., 2001;
Schedl et al., 1996;
Toy et al., 2002). We
analyzed, in both control (Pax6+/+) and Pax6 null mutants
(Pax6lacZ/lacZ)
(St-Onge et al., 1997), the
expression of the homeoprotein Crx, an essential photoreceptor determinant and
one of the earliest known exclusive markers for photoreceptor precursors
(PRPs) (Chen et al., 1997;
Freund et al., 1997;
Furukawa et al., 1997). For
the detection of amacrine precursors, we analyzed the expression of the
carbohydrate epitope VC1.1, which, during early embryogenesis, labels the
precursors of amacrine and horizontal cells
(Alexiades and Cepko, 1997;
Arimatsu et al., 1987;
Naegele and Barnstable,
1991). In E12.5 control retina, Crx (Fig. 1A,B) was detected in a few PRPs in the outer layers of the central OC, whereas VC1.1 (Fig. 1E,F) was observed in the inner layer of the central OC matching the location of ganglion and inner nuclear layer precursors. At a later stage (E15.5), in agreement with the central-to-peripheral progression of retinogenesis, their expression extended to the peripheral OC (Fig. 1C,D,G,H). At E12.5, the Pax6 protein was detected in most OC cells, including the VC1.1+ cells (Fig. 1E,F). However, its expression was barely detected in the Crx-expressing cells (Fig. 1B). On E15.5, Pax6 expression was absent from the Crx+ PRPs, but was low in the proliferative zone and high in the VC1.1+ cells of the inner nuclear layer (Fig. 1C,D,G,H). Taken together, these results indicated that, during normal retinal development, Pax6 is co-expressed with VC1.1 but is excluded from Crx-expressing cells.
We next characterized the expression of Crx and VC1.1 in the
Pax6lacZ/lacZ optic rudiment. Crx transcripts,
which are normally detected in only a few cells on E12.5 were detected in most
of the Pax6lacZ/lacZ OV neuroepithelium, including all
cellular layers (Fig. 1I,J).
This expanded expression domain of Crx was evident at later stages of
development (E15.5) (Fig.
1K,L), and it was not accompanied by misexpression of
photoreceptor-specific factors such as recoverin (data not shown)
(Haverkamp and Wassle, 2000;
Sharma and Ehinger, 1999). We
concluded that the Crx+ cells in the
Pax6lacZ/lacZ optic rudiment do not differentiate to
mature photoreceptors. These observations further indicate that neurogenesis
is abrogated in the Pax6-null OV
(Philips et al., 2005).
Notably, Crx was expressed in a highly heterogeneous fashion,
displaying high levels of expression in only a subset of cells of the
Pax6lacZ/lacZ OV neuroepithelium
(Fig. 1J,L).
|
). Thus, we tested the possibility of amacrine cell
genesis in the Pax6lacZ/lacZ OV by analyzing the
expression of VC1.1 (Alexiades and Cepko,
1997; Arimatsu et al.,
1987; Naegele and Barnstable,
1991) (Fig. 1K-L).
Interestingly, VC1.1 was detected in the Pax6lacZ/lacZ OV
neuroepithelium at E13 but not at E12.5, when it is normally expressed in the
control retina (data not shown) (Fig.
1E). Thus, its expression in the Pax6lacZ/lacZ
OV is delayed by 1 day relative to normal onset. The expression of VC1.1
persisted at later developmental stages in a subset of
Pax6lacZ/lacZ OV cells, indicating an initial commitment
of these cells to the amacrine cell lineage (E15.5)
(Fig. 1K,L). Moreover, the
VC1.1 epitope was not co-expressed in most of the Crx-expressing cells on
E15.5 (Fig. 1K,L), raising the
possibility of two distinct responses of the OV to Pax6 loss.
To further define the fate acquired by the
Pax6lacZ/lacZ OV cells, we characterized the expression of
the transcription factor Isl1, which is expressed by a subset of amacrine
cells (Galli-Resta et al.,
1997). In Pax6lacZ/lacZ, Isl1+
cells were detected in the optic rudiment, supporting initiation of the
amacrine differentiation pathway in the Pax6-mutant cells. Moreover, as
previously indicated (Philips et al.,
2005), at all of the tested stages, the
Pax6lacZ/lacZ OV neuroepithelium was deficient for markers
of other retinal cell types, such as the ganglion cell marker Pou4f2 and the
horizontal cell marker neurofilament Nf165
(Aramant et al., 1990;
Gan et al., 1996;
Xiang et al., 1993) (data not
shown). To further determine whether the VC1.1 and Isl1 cells identified in
the Pax6lacZ/lacZ OV give rise to mature amacrine
interneurons, we analyzed the expression of the selective amacrine cell marker
syntaxin and the pan-neural marker β-III tubulin
(Brandstatter et al., 1996).
Syntaxin and β-III tubulin were not detected in the
Pax6lacZ/lacZ OV, although in the normal retina their
expression was evident (E14, data not shown). These findings suggest that the
VC1.1 cells in the Pax6-null retina are amacrine precursors, which are unable
to differentiate to mature neurons.
Taken together, at the earliest stages preceding retinogenesis, Pax6 loss seems to expose two cryptic populations of RPCs: one seems to prematurely misexpress Crx, whereas in the other VC1.1 expression is delayed. Moreover, during early retinogenesis, Pax6 seems to be required for completion of neurogenesis: despite the apparent upregulation of the two early cell fate-specification programs to photoreceptors and amacrine cells, the Pax6-deficient OV progenitor populations were eventually abrogated in their capacity to terminally differentiate into mature neurons.
Divergent function of Pax6 within two distinct subsets of RPCs
The spatial and temporal roles of Pax6 were investigated by establishing
the Pax6flox allele (Materials and methods)
(Ashery-Padan et al., 2000).
Cre-mediated deletion of Pax6flox results in the
Pax6del allele (see Fig. S1 in the supplementary
material). Corresponding to the loss of Pax6 activity, the
Pax6del/del embryos exhibit the same phenotype as the
Pax6lacZ/lacZ mutants; developmental arrest at the OV
stage, premature misexpression of Crx, and delayed expression of the VC1.1
epitope (data not shown) (see Fig. S1 in the supplementary material).
In contrast to the differentiation arrest of the
Pax6lacZ/lacZ and Pax6del/del optic
rudiments, the selective removal of Pax6 from the OC after E10.5 resulted in
the generation of mature amacrine cells (see Fig. S3 in the supplementary
material) (Marquardt et al.,
2001). We therefore investigated the potential differences in the
requirement for Pax6 in early (OV) and later phase (OC) RPCs. To this
end, we analyzed the expression of VC1.1 and Crx, in control
(Pax6flox/flox) and
Pax6flox/flox;-Cre littermates
(Fig. 2). The region of Pax6
depletion in the Pax6flox/flox;-Cre OC was
determined by antibody labeling (Fig.
2A-H).
Interestingly, the expression of VC1.1 in the
Pax6flox/flox;-Cre OC was found to be
similar to its distribution in the control
(Fig. 2A): VC1.1 expression was
detected in the central OC and initially displayed little overlap with the
region of Pax6 inactivation (Fig.
2B). Thus, reminiscent of the situation in the
Pax6lacZ/lacZ OV, VC1.1 is not prematurely upregulated in
Pax6-deficient RPCs of the
Pax6flox/flox;-Cre retina. During
subsequent developmental stages, VC1.1 expression displayed a gradual
central-to-peripheral expansion in the
Pax6flox/flox;-Cre OC
(Fig. 2B,D,F), and was detected
in Pax6- cells (Fig.
2F, inset), although its expression was delayed in comparison with
that observed in the control retina (Fig.
2C,E,G).
|
-Cre mutants displayed
dramatic precocious upregulation of Crx expression
(Fig. 2J). This expression was
already detected on E12, i.e. about 48 hours prior to normal onset of Crx in
this region (Fig. 2K).
Moreover, in the Pax6flox/flox;-Cre
peripheral OC, the precocious Crx+ cells were localized
throughout the basal-apical extent of the retina
(Fig. 2J,L,N), as opposed to
the normal restriction of Crx expression to the PRPs located in the outer
layer of the OC (Fig.
2K,M,O).
In the peripheral Pax6flox/flox;-Cre
retina, most Pax6- cells expressed Crx by E12. By contrast, a
distinct population of Pax6- RPCs located toward the center of the
OC showed no detectable upregulation of Crx (compare
Fig. 2B,J, indicated zones `1'
and `2' and see diagram in Fig.
2). Quantitative analysis revealed that the proportion of
Crx+Pax6- cells (region 1) constituted 65%
(s.d.=9%) of the total number of Pax6-deficient cells on E12 (regions 1+2)
(Fig. 2Y). Moreover, we did not
detect any overlap between Crx expression and that of VC1.1 in Pax6-deficient
retinal cells (compare Fig.
2B,D,F,H with Fig.
2J,L,N,P). Thus, in striking similarity to the situation found in
the Pax6-deficient OV neuroepithelium of Pax6lacZ/lacZ
embryos, these data indicate the existence of two distinct progenitor
populations within the OC that differ in their requirement for Pax6 activity.
The first, more peripherally located population
(Fig. 2B,J, region 1)
precociously upregulates Crx following Pax6 inactivation, whereas the second
(Fig. 2B,J, region 2),
centrally located population does not display Crx expression following loss of
Pax6.
|
-Cre OC. At E14, the
proportion of Crx+Pax6- cells in regions 1 and
2 was reduced to 60% (s.d.=4%) (Fig.
2Y). By E16, this proportion dropped further to 40% (s.d.=10%)
(Fig. 2Y). Eventually, on E18,
the Crx+Pax6- cells constituted only a very
small portion of the total number of Pax6- cells in the peripheral
Pax6flox/flox;-Cre OC
(Fig. 2P), whereas VC1.1 and
syntaxin (Fig. 2H,X) were
detected in many cells at the retinal periphery and their expression extended
across the apical-basal axis of the OC, although normally these markers are
detected in the inner layers, corresponding to the location of amacrine cells
(Fig. 2G,W). Some cells in the
peripheral Pax6flox/flox;-Cre OC were
negative for both VC1.1 and syntaxin on E18
(Fig. 2H,X). At postnatal
stages, Crx was not detected in the Pax6- deficient OC
periphery, whereas the Pax6- cells expressed syntaxin
within the peripheral retina, in accordance with their eventual
differentiation into amacrine interneurons (see Fig. S3 in the supplementary
material) (Marquardt et al.,
2001).
Overall, the peripheral
Pax6flox/flox;-Cre retina displayed a
markedly reduced size relative to the control retina, in agreement with the
reduced mitotic rate of the Pax6- deficient RPCs
(Marquardt et al., 2001). This
raised the possibility that the observed shift in the relative proportion of
region 1 and 2 progenitors in the
Pax6flox/flox;-Cre was due to differential
impacts of Pax6 deficiency on the mitotic rates of the distinct progenitor
populations. Alternatively, loss of Pax6 may also differentially affect the
subsequent survival of region 1 and 2 cells. To address these possibilities,
we performed quantitative analysis of cell proliferation in
Pax6flox/flox;-Cre and control retinas
through BrdU pulse chase assays on E12-16
(Fig. 2Q-V,Z). Following a 1.5
hours BrdU pulse, at E12, both region 1 and 2 progenitors in the
Pax6flox/flox;-Cre retina displayed
similarly reduced BrdU incorporation relative to control peripheral retinas
(14.7%, s.d.=2.1% and 16.9%, s.d.=2% BrdU+ cells, respectively,
versus 25.5%, s.d.=3.6% in the control)
(Fig. 2Z). However, a marked
difference in the relative incorporation of BrdU was detected in the E14
Pax6flox/flox;-Cre retina, with only 10.3%
BrdU+ (s.d.=2.6%) cells in region 1, compared with 19.3% (s.d.=1%)
BrdU+ cells in region 2, and 23.8% (SD=1.8%) in the control
peripheral retina (Fig. 2Z).
These results indicated that in the
Pax6flox/flox;-Cre retina, the marked
expansion of region 2 progenitors relative to region 1 is due to the different
impacts of loss of Pax6 activity on their proliferation.
To address whether differential rates of apoptosis may additionally account
for the observed relative shifts in OC population sizes, we performed
immunodetection of cleaved caspase 3 (Ccaspase 3) in
Pax6flox/flox;-Cre and control retinas
(Di Cunto et al., 2000). We
did not detect any significant increase in the number of Ccaspase
3+ cells in the
Pax6flox/flox;-Cre compared with the
control OCs at E14 and E16 (data not shown). This indicates that the observed
relative shifts in OC population sizes are due to differences in mitotic rate,
rather than to selective elimination through apoptosis. The proliferation
arrest during early embryogenesis of region 1 cells and some cell loss due to
apoptosis, are consistent with the eventual elimination of
Pax6-;Crx+ cells
(Fig. 2P; see Fig. S3 in the
supplementary material)
Pax6 controls different neurogenic programs in region 1 and region 2 RPCs
Previous data have indicated that the retinal expression of a number of
proneural bHLH factors depends on Pax6 function
(Marquardt et al., 2001;
Scardigli et al., 2003). We
therefore further investigated the impact of Pax6 inactivation on the
expression of selected bHLH factors in both region 1
(Pax6-Crx+) and region 2
(Pax6-Crx-) pools. This analysis was performed
on E15, when the expression of most bHLH factors and Crx has progressed into
the OC periphery corresponding to regions 1 and 2
(Fig. 3A-E; see Fig. S2 in the
supplementary material). The region of Pax6 loss was determined by detection
of Pax6 with antibodies or by monitoring the expression of hAP activity in the
Pax6flox/flox;-Cre;Z/AP OCs
(Fig. 3; see Fig. S2 in the
supplementary material). In the
Pax6flox/flox;-Cre embryos, Crx
misexpression was detected only in the peripheral compartment of the
Pax6-depleted OC (Fig. 3F; see
Fig. S2 in the supplementary material), whereas Atoh4 expression was abolished
in all of the Pax6-depleted compartments, in agreement with previous reports
on the direct regulation of Atoh4 by Pax6
(Fig. 3G; see Fig. S2 in the
supplementary material) (Marquardt et al.,
2001; Scardigli et al.,
2003). We next compared the expression pattern of additional
proneural bHLH factors in the two Pax6 mutant regions: region 1, which is
defined as the Pax6-Atoh4-Crx+
domain (Fig. 3F,G); and the
Pax6-Atoh4-Crx-demarcated region 2
(Fig. 3F,G). On adjacent
sections, analysis of the expression of Atoh3, Neurod1 and
Atoh7 revealed their downregulation in the
Pax6flox/flox;-Cre peripheral OC
(Fig. 3H-J). The normal
expression of Atoh3, in the photoreceptor layer
(Fig. 3C), was virtually
extinguished from both region 1 and region 2 progenitors
(Fig. 3H), thus resembling the
loss of Atoh4 from the Pax6-deficient cells
(Fig. 3G). Interestingly, the
expression of Neurod1 appeared to be differentially affected in
regions 1 and 2 of Pax6flox/flox;-Cre
mutants, being severely diminished in region 1 progenitors. At the same time,
low levels of Neurod1 expression were maintained in region 2, similar
to its expression in the neuroblast layer of the control retina (compare
Fig. 3D with
Fig. 3I). However, the
characteristically high levels of Neurod1 expression in the
presumptive photoreceptor-containing outer nuclear layer was severely
diminished in both regions 1 and 2 of the
Pax6flox/flox;-Cre
(Fig. 3I). Similar to the
neuroblast layer expression of Neurod1, Atoh7 mRNA levels displayed
marked differences between regions 1 and 2 of the
Pax6flox/flox;-Cre. Whereas Atoh7
expression was almost completely lost from region 1 RPCs, region 2 RPCs
displayed persistent, albeit reduced levels of Atoh7 mRNA compared
with the peripheral control retina (compare
Fig. 3E with
Fig. 3J). Retinal cell fate
depends on the combination of bHLH factors expressed within the cells; for
example, Neurod1 has previously been implicated in controlling
amacrine cell genesis (Inoue et al.,
2002; Ohsawa and Kageyama,
2008). The persistent Atoh7 and Neurod1
expression in Pax6-Crx- region 2 RPCs thus
suggests the funneling of these progenitors towards an amacrine fate through
loss of most of the other essential neurogenic programs
(Marquardt et al., 2001).
Taken together, these data indicate that Pax6 controls different sets of
neurogenic programs in two inherently distinct subsets of OC progenitors.
|
-Cre retina suggested
premature acquisition of the photoreceptor cell fate by these progenitor
cells. To further test this idea, we investigated the expression of a number
of factors associated with the photoreceptor-differentiation pathway in the
Pax6lacZ/lacZ and
Pax6flox/flox;-Cre;Z/AP. In the latter
model, the region of Pax6 inactivation was monitored by detection of hAP
expression from the Z/AP-transgene (Fig.
4J). The paired-like homeodomain protein Rx is one of the earliest
known RPC markers, is essential for initiating retinal development and has
been implicated in eventual regulation of photoreceptor-specific gene
expression (Bailey et al.,
2004; Kimura et al.,
2000; Wang et al.,
2004). In both the Pax6lacZ/lacZ OV and the
Pax6flox/flox;-Cre;Z/AP mutants, the
expression of Rx was maintained at levels similar to the
stage-matched control retina (not shown)
(Baumer et al., 2003;
Marquardt et al., 2001). In
contrast to Rx, the expression of Otx2, a member of the
otd/Otx gene family essential for photoreceptor differentiation
(Fig. 4B)
(Nishida et al., 2003), was
virtually undetectable in the Pax6-deficient RPCs of
Pax6flox/flox;-Cre;Z/AP mutants
(Fig. 4H), as well as in the
distal neuroretinal portion of the Pax6lacZ/lacZ OV
(Fig. 4E). Similarly, the
expression of Trb2 (thyroid hormone receptor β 2), which
regulates M and S opsin expression in cones and is expressed in the PRPs layer
on E15 (Fig. 4C)
(Applebury et al., 2007;
Ng et al., 2001;
Shibusawa et al., 2003), was
absent from Pax6lacZ/lacZ OVs
(Fig. 4F) and from both region
1 and 2 progenitors in
Pax6flox/flox;-Cre;Z/AP mutants
(Fig. 4I), despite the
detection of high levels of Crx RNA in region 1 cells on adjacent
sections of the same specimen (Fig.
4G). Together, these data indicate that the
Pax6-Crx+ region 1 progenitors in the
Pax6flox/flox;-Cre retina initiate an
early photoreceptor specification program, but eventually fail to enter
terminal differentiation towards mature photoreceptor neurons.
Pax6 binds the Crx promoter in the embryonic mouse retina
The dramatic change in Crx expression in both
Pax6lacZ/lacZ and
Pax6flox/flox;-Cre mutants, together with
the early appearance of Crx close to the onset of Pax6 inactivation, suggested
direct inhibition of Crx expression by Pax6 in a subpopulation of RPCs. A 2 kb
region has been shown to contain crucial regulatory sequences required for
full expression of Crx in the developing retina
(Furukawa et al., 2002). The
300 bp sequence adjacent to the transcription start site is conserved among
mammals (81% conservation between mice and humans). In addition, this region
includes putative binding sites for paired-type homeodomain-containing
proteins (Nishida et al.,
2003; Tatusova and Madden,
1999). To establish whether Pax6 binds directly to the proximal
Crx promoter in vivo, we performed a ChIP analysis using a specific
antibody against Pax6 to immunoprecipitate chromatin from embryonic (E13)
mouse eyes (Fig. 4K). The
sequences of the Crx promoter were amplified from the
immunoprecipitated chromatin by PCR. We also used the Crx 3'
UTR sequences and Optimidin intron 6 sequences as reference regions
(Grinchuk et al., 2005). In
the chromatin prepared from E13 retina, Pax6 was found to occupy the
Crx promoter region but not its 3' UTR or the
Optimidin intron 6 sequences (Fig.
4K, not shown). Binding of Pax6 to Crx promoter was not identified
in the limb chromatin where Pax6 is not expressed, and the binding was not
detected with non-specific IgG (Fig.
4K). In addition, we tested one putative binding site for Pax6
[chr7:16465201-16465450; predicted by MatInspector
(Cartharius et al., 2005)] but
this site did not bind in vitro to Pax6 by electromobility shift assay (EMSA;
data not shown). We therefore conclude that Pax6 interacts directly with the
Crx promoter region in the embryonic retina, and that Pax6 activity on the Crx
promoter possibly requires additional co-factors or occurs at different
Pax6-binding site than the one indicated by the in silico prediction. The ChIP
data combined with gene ablation studies suggest direct, albeit
context-dependent, regulation of Crx by Pax6 in retinal progenitor cells.
|
-Cre transgene mediates recombination within the OC
periphery, including retinal progenitors and non-neuronal progenitors that are
destined to iris and cilliary body fates
(Davis-Silberman and Ashery-Padan,
2008; Marquardt et al.,
2001). The misexpression of Crx, which is observed in the most
peripheral OC, may therefore represent a unique role for Pax6 in the
population of non-neuronal progenitors rather than a novel function in retinal
neurogenesis. To explore this possibility, we employed the
Chx10-Cre-transgenic mouse line
(Rowan and Cepko, 2004). The
recombination pattern mediated by this transgene is a mosaic, yet it overlaps
with Chx10 expression domains; at mid-gestation (E14), the
Chx10-Cre recombination has been reported to occur in the OC in
patches of neuronal precursors but to be excluded from the most distal tips
where the non-neuronal progenitors reside
(Rowan and Cepko, 2004). We
characterized the phenotype of the Pax6flox/flox;Chx10-Cre
OC on E14. At this stage, Pax6 is normally detected in most cells of the OC;
however, it is reduced to almost undetectable levels in the Crx-expressing
PRPs (Fig. 5A,B). In the
Pax6flox/flox;Chx10-Cre mutants, the recombination pattern
was monitored by detection of Pax6 protein loss
(Fig. 5C-E). Corresponding with
the reported pattern of Chx10-Cre activity, Pax6 was depleted from
patches of RPCs in both the distal and proximal OC, but its expression was
maintained in the most distal non-neuronal progenitors
(Fig. 5C). In some of the
Pax6-depleted regions, Crx misexpression was detected across the apical-basal
OC; however, there were patches of Pax6-deficient cells that did not
upregulate Crx (Fig. 5C,D,
white arrowheads). At E14, however, most of the
Pax6-Crx- cells had not yet upregulated VC1.1 expression
(data not shown), corresponding with the delayed expression of VC1.1 observed
in the Pax6flox/flox;-Cre mutants
(Fig. 2D). However, the
expression of VC1.1 was evident in Pax6- cells of the
Pax6flox/flox;Chx10-Cre mutants on E16, whereas in the
normal retina at this stage, all of the VC1.1 cells co-expressed Pax6
(Fig. 5F,H). Moreover, there
was no overlap in the expressions of VC1.1 and Crx in the
Pax6flox/flox;Chx10-Cre mutants, similar to their separate
distribution in the control and Pax6flox/flox;a-Cre
mutants (Fig. 5G,I;
Fig. 2F,N).
|
-Cre OC following loss of
Pax6 were identified in the Pax6flox/flox;Chx10-Cre
mutants (Pax6-Crx+ and Pax6-Crx-
VC1.1+), although the recombination mediated by Chx10-Cre
did not include the non-neuronal progenitors of the OC. We therefore conclude
that the two phenotypes observed in the OC following Pax6 loss reflect the
distinct roles of Pax6 in the RPCs. Notably, the
Pax6-Crx+ cells were detected both adjacent to, and at a
distance from, non-recombined Pax6-expressing cells
(Fig. 5C-E). This demonstrates
that normal cells do not inhibit Crx misexpression in adjacent mutant cells
and provides further support for the notion that the two distinct phenotypes
of the Pax6-deficient OC represent different intrinsic requirements for Pax6
in the developing retina.
To determine the eventual phenotype of Pax6-deficient RPCs in the
Pax6flox/flox;Chx10-Cre mutants, we traced the mutant
cells with Z/AP and determined their neuronal phenotype by
co-labeling with antibodies to the amacrine-specific marker syntaxin or the
photoreceptor determinant, recoverin. The phenotype of the hAP+
cells in the Pax6flox/flox;Chx10-Cre;Z/AP retina was
similar to that observed in the
Pax6flox/flox;-Cre mice (see Fig. S3 in
the supplementary material) (Marquardt et
al., 2001). In regions where hAP was detected across the retina,
thus originating from Pax6-deficient RPCs, the laminar organization was lost
and most cells co-expressed hAP and syntaxin, but not recoverin (see Fig. S3
in the supplementary material). This further demonstrates that, regardless of
the location of Pax6-deficient RPCs in the central or peripheral OC, the
Pax6-deficient RPCs that maintain the differentiation potential are eventually
restricted in their differentiation capacity and differentiate exclusively
into amacrine interneurons.
In the Pax6flox/flox;-Cre, the
Pax6-Crx- cells were consistently localized toward the
center of the OC, whereas the Pax6-Crx+ cells were
identified more distally (Fig.
2). We therefore asked whether the central or peripheral position
of the mutated cells in Pax6flox/flox;Chx10-Cre is
predictive of their eventual phenotype
(Pax6-Crx- or
Pax6-Crx+). The proportion of the
Pax6-Crx+ area relative to the total
Pax6-deficient area was measured in the peripheral and central thirds of the
OCs and the average values were calculated. In the peripheral third of the OC,
79% (s.d.=18%) (Fig. 5J) of the
Pax6-deficient regions were Crx+, while in the central
third, only 34% (s.d.=10%) (Fig.
5J) of the Pax6-deficient domain misexpressed Crx; this difference
was highly significant (Fig.
5J). Thus, the phenotypic outcome of Pax6-deficient RPCs
correlated with the location of the cells within the OC; mutation in Pax6 in
the peripheral OC is most likely to result in misexpression of Crx, whereas
Pax6 deletion more centrally is likely to result in differentiation of the
Pax6-deficient cells to amacrine interneurons.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
An early Pax6-independent subdivision of RPCs into distinct progenitor pools
In Pax6-null mutants, the retinal neuroepithelium of the OV
rudiment displayed two distinct subsets of progenitors that differed in their
phenotype: in one population, premature upregulation of Crx was observed,
whereas in the other, Crx was not expressed and the appearance of VC1.1 was
delayed. The detection of two distinct phenotypes in the Pax6 null
optic-rudiment indicated a prior distinction of discrete RPC subsets, well
before the normal onset of retinal cell differentiation and that these
progenitor populations emerge independently of Pax6 activity during early
stages of retinogenesis.
Previous data have indicated that the early subdivision of the OV
neuroepithelium into spatially separate optic stalk, neuroretinal and pigment
epithelial progenitor fields requires the activity of signaling pathways such
as Shh from the midline, TGFβ signaling from the extra-ocular mesenchyme
and Fgfs from the surface ectoderm
(Fuhrmann et al., 2000;
Macdonald and Wilson, 1997;
Nguyen and Arnheiter, 2000).
The spatial distinction into these principal progenitor domains was found to
be maintained in the OV of Pax6-null mutants, but was lost upon
elimination of both Pax6 and Pax2
(Baumer et al., 2003;
Grindley et al., 1995). Here,
we found that the early elimination of Pax6 in the
Pax6lacZ/lacZ mutant leads to a loss of spatial separation
between regions 1 and 2 RPCs within the presumptive neuroretinal domain. Both
the Crx+VC1.1- and the
Crx-VC1.1+ RPC pools were found to be
intermixed within the Pax6lacZ/lacZ OV, in contrast to
their spatial separation in the
Pax6flox/flox;-Cre and
Pax6flox/flox;Chx10-Cre retinas. The present study thus
suggests that early patterning of the OV and formation of the OC are
accompanied by the establishment of distinct subpopulations of RPCs within the
neuroretina. The dependency on Pax6 for this regionalization of the RPCs
during early stages of eye development may directly relate to the function of
Pax6 in the OV, or it may reflect a secondary outcome of the arrest in OC
formation or absence of the lens, which has been shown in previous studies to
be required for the morphology of the OC
(Ashery-Padan et al., 2000).
Together, the establishment of regional distinctions between RPCs along the
proximodistal axis of the neuroretina appears to depend, directly or
indirectly, on an early phase of Pax6 activity - a dependency that ceases once
the optic-cup stages are reached.
Dual requirements for Pax6 within the two subpopulations of RPCs
Recent studies have shown that in the developing neocortex there are
several distinct neurogenic progenitor cells that are multipotent, including
the radial glia and intermediate progenitor cells
(Hevner, 2006;
Pontious et al., 2008). Within
these populations, Pax6 is expressed and plays different roles, depending on
the temporal and spatial context
(Guillemot, 2005;
Pinto and Gotz, 2007;
Warren et al., 1999).
Moreover, a dual role for Pax6 has been reported in the generation of neurons
of the adult olfactory bulb, where Pax6 was found to initially regulate the
establishment of the neuronal lineages and, subsequently, their specification
toward a periglumerular cell fate (Hack et
al., 2005). In contrast to the developing neocortex, differences
among RPCs have not yet been recognized in the developing retina. However,
there are several lines of evidence supporting distinct transient states of
these cells: first, considering the central-peripheral pattern of
differentiation, it is likely that the RPCs located adjacent to
differentiating neurons at the central OC are exposed to different cues from
the RPCs located far from the differentiation front, at the OC periphery.
Second, recent findings have shown the differential expression of genes in the
central versus peripheral regions (Adler
and Canto-Soler, 2007; Koso et
al., 2006; Koso et al.,
2007). Finally, in this study, two distinct phenotypes of RPCs
were identified after Pax6 inactivation in the OC, including differences in
the expression Crx, the expression profile of proneural bHLH genes,
proliferation index and neurogenic potential. Moreover, these different
phenotypes were correlated to the location of the cells along the
central-peripheral axis of the OC. Together, these findings indicate an
important distinction between Pax6 activities within adjacent RPC pools,
suggesting an inherent difference between RPC populations. Considering that
all retinal cell types eventually populate both central and peripheral retina
in the adult, it seems likely that the differences documented here between
central and peripheral OC RPCs primarily reflect distinct differentiation
stages of the multipotent progenitor pools, similar to the transient states
observed in cortical neurogenesis (Hevner,
2006; Pontious et al.,
2008) rather than differences in cell specification. In this case,
the role of Pax6 is to promote the maturation of progenitor cells and their
eventual differentiation to all of the retinal cell types.
Analogous to the early intrinsic differences identified here between distal
and proximal RPCs, recent studies have found a regional distribution of
components of the Wnt, Hedgehog, BMP and Notch signaling pathways along the
proximal-distal axis of the OC (Adler and
Canto-Soler, 2007; Yaron et
al., 2006). Similarly, the stem-cell epitope CD15 was found to be
transiently expressed in a Wnt-dependent manner within a subset of RPCs
located at the retinal periphery (Koso et
al., 2007). These factors may therefore create focal differences
among RPCs and underlie the intrinsic differences that were exposed here
following loss of Pax6, although their precise role and their
regulatory relationship with Pax6 remain to be addressed.
Involvement of Pax6 in the transcriptional network regulating photoreceptor differentiation in mammals
The iterative deployment of Pax6 in the process of eye formation in
evolutionarily distant organisms, suggests that there are common
transcriptional targets for Pax6 in the different species, such as the
regulation of opsin gene expression (Arendt
et al., 2004; Zuker,
1994; Gehring,
2005). In support of this idea, eyeless, the fly homolog
of Pax6, was found to be expressed in photoreceptors and was
subsequently shown to regulate the expression of the Drosophila
rhodopsin genes in these cells (Papatsenko
et al., 2001; Quiring et al.,
1994; Sheng et al.,
1997). In vertebrates, however, this role for Pax6 does not appear
to be conserved, in line with the rapid downregulation of Pax6 expression in
differentiating photoreceptors during vertebrate retinogenesis. Moreover, our
ChIP data indicate selective binding of Pax6 protein to the Crx
promoter region, supporting its role as a direct transcriptional repressor of
photoreceptor fate. The current study reveals the complex involvement of Pax6
in the transcriptional network leading to photoreceptor differentiation in
mammals (Fig. 6). Surprisingly,
although in both regions Pax6 is essential for completion of the
photoreceptor-differentiation program, its regulation of the genes involved in
the photoreceptor lineage is different in the two regions of the OC: in region
1 it plays a role in inhibiting the onset of Crx expression, whereas in region
2 it is required for the expression of Crx. Thus, based on these findings, the
ancestral role of Pax6 in regulating opsin expression appears to have switched
to a different, more complex, level of control over key retinogenic
programs.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/24/4037/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
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