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First published online 1 March 2006
doi: 10.1242/dev.02311
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1 Sackler Faculty of Medicine, Department of Human Molecular Genetics and
Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel.
2 The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010
North Torrey Pines Road, La Jolla, CA 92037, USA.
3 The Howe Laboratory, Harvard Medical School, Boston, MA 02114, USA.
Author for correspondence (e-mail:
ruthash{at}post.tau.ac.il)
Accepted 6 February 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Notch1, Cre/loxP, Lineage tracing, Retinal progenitor cells, Photoreceptors, Mouse, Notch1, Retina development
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Despite vast
differences in the total time scale, the relative histogenic birth order of
the different retinal cell types is largely conserved among vertebrate
species, suggesting that the same principal mechanisms mediate the
differentiation program for the different retinal cell types.
Cell-lineage studies in vertebrates have revealed that RPCs are multipotent
progenitors, as each progenitor may give rise to each of the retinal cell
types (Turner and Cepko, 1987;
Wetts and Fraser, 1988).
However, heterochronic culturing experiments suggest that the differentiation
potential of the progenitors is dynamic and changes as development progresses
(Cepko et al., 1996;
Livesey and Cepko, 2001;
Watanabe and Raff, 1990). It
has been proposed that the differentiation competence of the progenitors is
determined by a combination of intrinsic and extrinsic factors
(Livesey and Cepko, 2001;
Marquardt, 2003). The
intrinsic signals include a combination of factors, such as bHLH- and
homeodomain-containing transcription regulators
(Kageyama et al., 1997;
Levine et al., 2000;
Marquardt, 2003). Studies have
indicated that several secreted factors, including sonic hedgehog, GDF11,
retinoic acid (RA) and taurine, play a role in cell-type specification in the
retina (Kim et al., 2005;
Levine et al., 2000;
Neumann and Nuesslein-Volhard,
2000). In addition to the influence of secreted factors, another
important developmental cue is cell-cell contact-dependent mechanisms, such as
those mediated by Notch signaling. The Notch pathway has been shown to
function in a variety of processes regulating tissue growth, patterning and
morphogenesis in different organisms, including insects and mammals
(Artavanis-Tsakonas et al.,
1999; Kopan and Cagan,
1997).
The Notch family members are transmembrane receptors with large
extracellular and intracellular domains connected by a single
membrane-spanning domain (del Amo et al.,
1993). The current model for Notch signaling suggests that upon
binding of the Notch receptor to its membrane-linked ligands, the receptor is
proteolyzed and the released intercellular domain operates as a transcription
regulator through interaction with nuclear mediators such as RBP-jk
(Lai, 2004). Activation of the
Notch receptor upregulates the expression of target genes, such as
Hes1/Hes5 in mammals (Ohtsuka et
al., 1999). These genes negatively regulate the expression of
proneural bHLH transcription factors that are known to affect cell-fate
specification of progenitors (Kageyama et
al., 1997). Thus, in cells in which Notch signaling has been
initiated, differentiation is generally inhibited.
Current studies on flies and vertebrates suggest that the role of Notch is
not uniform but depends on the developmental and cellular context of the
tissue (Bray, 1998;
Perron and Harris, 2000). In
vertebrate neurogenesis, Notch1 and its ligand Delta1 have been implicated in
maintaining a pool of uncommitted precursors, while release from Notch1/Delta1
inhibition allows the cells to differentiate
(Chitnis et al., 1995;
Henrique et al., 1995;
Henrique et al., 1997;
Perron and Harris, 2000). In
addition to this generalized role in preventing the premature depletion of the
progenitor pool, several studies suggest that Notch-mediated lateral
inhibition might inhibit the acquisition of specific cell fates more
selectively, in particular that of the ganglion cells. This was suggested, for
example, following antisense treatment of chick retina, which resulted in an
increased number of ganglion cells (Austin
et al., 1995; Waid and McLoon,
1998). Such an effect on cell fate due to Notch1 reduction is more
pronounced at early stages and seems to be gradually lost during later stages
of retinal development (Silva et al.,
2003), indicating that differences in the intrinsic makeup of the
progenitors determine their eventual response to Notch1 reduction. The most
direct evidence for the role of Notch in cell-type specification in the retina
was obtained from a study of mice deficient in one of the Notch1 effectors,
Hes1. Inactivation of Hes1 resulted in premature differentiation into rods
while bipolar cells failed to survive
(Tomita et al., 1996). Other
studies, however, support the role of Notch1 in vertebrate gliogenesis. In
particular, Notch1 and its effectors, Hes1 and Hes5, seem to promote certain
glial fates, such as Müller glia in the retina
(Chambers et al., 2001;
Dorsky et al., 1995;
Furukawa et al., 2000;
Gaiano and Fishell, 2002;
Hojo et al., 2000;
Scheer et al., 2001). By
contrast, Notch1 has been found to inhibit gliogenesis in the optic nerve
(Wang et al., 1998) and spinal
cord (Genoud et al., 2002).
These seemingly conflicting observations may reflect a significant influence
on Notch1 activity of cell-intrinsic changes that depend on the respective
cellular and developmental context.
The direct in vivo study of the role of Notch1 in mice was previously
precluded owing to the early embryonic lethality of Notch1-deficient embryos
just when neuronal differentiation is initiated
(Conlon et al., 1995;
Swiatek et al., 1994). Thus,
the precise requirement of Notch1 activity during different stages of
vertebrate neurogenesis still remains largely elusive. Here we used the
Cre/loxP approach to inactivate the Notch1 gene exclusively
in retinal progenitor cells (RPCs) prior to the onset of their
differentiation. This somatic inactivation resulted in abnormal retinal
morphology and a reduction in eye size. Similar to the known activity of Notch
signaling in invertebrates, Notch1 inactivation in RPCs resulted in the
dramatic downregulation of Hes5 and Hes1, and upregulation of several
proneural transcription factors, eventually triggering premature
differentiation of the RPCs. Surprisingly, a dramatic increase in the number
of cone-photoreceptor precursors was detected in the
Notch1- retina at the expense of other early- and
late-born retinal cell types. These Notch1- photoreceptor
precursors followed the pattern of cone-photoreceptor differentiation and
developed to cone photoreceptors postnatally. Thus, in addition to its
well-established role as a generic inhibitor of neuronal differentiation, our
results demonstrate an essential requirement for Notch1 activity in
facilitating the diversification of retinal cell types by selectively
suppressing the acquisition of the cone-photoreceptor cell fate.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-Cre transgenic
line (Fig. 1)
(Radtke et al., 1999;
Marquardt et al., 2001). For
cell-lineage tracing of Cre expressing cells the Z/AP reporter line
was employed (Fig. 1)
(Lobe et al., 1999).
Histology, immunofluorescent-analysis, BrdU and TUNEL assays
Tissues were fixed for 2 hours or overnight in 4% paraformaldehyde (PFA) at
4°C, dehydrated and embedded in paraffin wax. Paraffin sections (5-10
µm) were stained with Hematoxylin and Eosin (H&E) using standard
procedures. Human placental alkaline phosphatase (AP) staining was performed
on sections as previously described (Lobe
et al., 1999). Immunofluorescent analysis was performed on dewaxed
paraffin sections as previously described
(Ashery-Padan et al., 2000).
The primary antibodies were: rabbit anti-Pax6 (1:1000, Chemicon), goat
anti-Brn3b (1:100, Santa Cruz), goat anti-AP (1:100, Santa Cruz), mouse
anti-Nf165 (1:500, Hybridoma Bank), rabbit anti-PKC (1:1000, Santa
Cruz), rabbit anti-CycD3 (1:100, Santa Cruz), mouse anti-Syntaxin (1:600,
Sigma), rabbit anti-recoverin (1:750, kind gift from C. Koch), rabbit
anti-S-opsin (1:1000), mouse anti-rhodopsin (1:1000)
(Applebury et al., 2000) and
rabbit anti-Hes1 (1:200) (Lee et al.,
2005). Secondary antibodies conjugated to rhodamine red-X or Cy2
were from Jackson Immuno Research Laboratories. For detection of cones, peanut
agglutinin (PNA) conjugated to fluorescein (1:200, Vector) was employed
(Blanks and Johnson, 1983).
For detection of cells in the S phase, BrdU (10 µl/g of 14 mg/ml) was
injected 1.5 hours before the sacrifice. Antibody staining using mouse
anti-BrdU (1:100, Chemicon) was performed as described
(Marquardt et al., 2001).
TUNEL assay was performed using the in situ Cell Death Detection Kit (Roche).
Slides were viewed with an Olympus BX61 fluorescent microscope or
laser-scanning confocal microscope CLSM 410 (Zeiss) and images were analyzed
using the image analysis system `AnalySIS'.
The relative proportion of Brn3b, TUNEL, BrdU, Crx, cone-arrestin, Gnat1
and recoverin positive cells was calculated from the total nuclear
(DAPI+) area. The areas were measured using the color-threshold
function of `AnalySIS' software on the same predefined region of the
peripheral optic cup for the
Notch1f/f;-Cre and the corresponding
control eyes. The measurements were conducted on serial sections (
50
µm apart) with well-preserved morphology and that were central based on the
detection of the lens. For each eye, an average value for the presented
parameter was calculated from all sections. The presented ratio values are the
averages obtained from all eyes (n) with the same genotype.
Analysis of Brn3 expression and AP detection on dissociated embryonic retinas
E14 retinas were dissected and incubated for 1 hour at room temperature in
dissociation solution (125 mM NaCl, 2 mM KCl, 1.2 mM EDTA, 5 mM HEPES pH 7.4),
then triturated to a single-cell suspension and fixed 15 minutes on
poly-D-lysine-coated glass slides with 4% PFA. The cells were subjected to
immunofluorescent-analysis (as above) using Brn3b specific antibody for the
detection of ganglion cells. After documenting Brn3b expression in random
fields, AP activity was detected on the same slides as described
(Lobe et al., 1999). The AP
images were superimposed on the images of the Brn3 antibody staining and the
ratio of Brn3b+ to AP+ cells was calculated.
In situ hybridization
In situ hybridization analysis was performed on dewaxed paraffin sections.
Hybridization was conducted overnight at 55°C with digoxigenin-labeled
probes (3 µg/ml). The slides were then treated with RNaseA, washed, blocked
with 10% normal goat serum (NGS) and incubated with sheep anti-digoxigenin Fab
fragments conjugated to alkaline phosphatase (1:2500, Roche) in PBST with 1%
NGS overnight at 4°C, washed and incubated in BM Purple (Roche). The
probes used in this study were as follows: Notch1
(Schroder and Gossler, 2002),
Hes5 (Ohtsuka et al., 1999),
Neurod1 (Lee et al., 1995),
Ngn2 (Sommer et al., 1996),
Math5 (Brown et al., 1998),
Mash1 (Lo et al., 1997), Math3
(Akagi et al., 2004), Nr2e3
(Chen et al., 2005), Otx2
(Nishida et al., 2003), Crx
(Furukawa et al., 1997) and
Thrß2 (Ng et al., 2001;
Wood et al., 1994).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-Cre mouse lines (Fig.
1A,B) (Marquardt et al.,
2001; Radtke et al.,
1999). Notch1f has been shown to render Notch1
inactive following Cre-mediated recombination
(Fig. 1A)
(Radtke et al., 1999), while
in the -Cre transgenic line, Cre expression is restricted to
the RPCs residing in the temporal and nasal parts of the peripheral optic cup
(Kammandel et al., 1999;
Marquardt et al., 2001). To
trace Cre activity in vivo, we employed the Z/AP reporter mouse line
(Lobe et al., 1999). In this
line, the Cre-mediated recombination enables the expression of the AP
reporter gene (Fig. 1C,
Fig. 2I)
(Lobe et al., 1999).
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-Cre
inactivates Notch1, we monitored AP activity
(Fig. 1D,I) in
-Cre;Z/AP control retina sections at E12.5
(Fig. 1D-G) and E13.5
(Fig. 1I-L). In addition, the
expression patterns of Notch1
(Fig. 1E,J), its downstream
target Hes5 (Fig.
1F,K) and the earliest ganglion cell marker Brn3b
(Fig. 1G,L) were monitored on
adjacent sections by in situ hybridization
(Fig. 1E,F,J,K) or antibody
labeling (Fig. 1G,L). The
results are summarized schematically (Fig.
1H,M) and demonstrate that at E12.5, Notch1
(Fig. 1E) and Hes5
(Fig. 1F) transcripts are
already present in most of the optic cup, including a part in the peripheral
retina overlapping the domain of -Cre activity
(Fig. 1H). At this stage, the
expression of Notch1 and Hes5 seems to precede the central
to peripheral differentiation wave, as Brn3b expression is more central to the
Notch1, Hes5 and AP-expressing cells
(Fig. 1G,H). At E13.5
(Fig. 1I-M), Notch1,
Hes5 and Brn3b expression overlaps with AP in the peripheral retina,
excluding the most peripheral tip of the optic cup where only AP is detected
and later gives rise to the non-neuronal ocular tissues. This analysis
suggests that the -Cre-mediated recombination will delete
Notch1f in the RPCs located in the peripheral optic cup
prior to the onset of cell differentiation.
Notch1 inactivation results in microphthalmia and distorted retina morphology
Microphthalmia was observed in the
Notch1f/f;-Cre mice (P60,
Fig. 2E). To characterize the
morphology of the Notch1f/f;-Cre eyes,
detailed histological analysis was conducted by Hematoxylin-Eosin (H&E)
staining of eye sections from control littermate controls
(Fig. 2B-D) and
Notch1f/f;-Cre mutants
(Fig. 2F-H). In the control
eyes at P15, the cellular and synaptic layers of the retina have formed
(Fig. 2B). In the
Notch1f/f;-Cre eyes, however, distortion
of the retinal morphology, including large rosettes and disruption of the
retinal layers, was observed (Fig.
2F), and this aberrant morphology was already prominent at E17.5
(Fig. 2G). Further histological
analysis revealed that the rosettes are first apparent at E13.5 in the
peripheral retina of Notch1f/f;-Cre;Z/AP
embryos (Fig. 2H). Importantly,
we could not detect any noticeable phenotype in the retina of the
Notch1f/+;-Cre and
Notch1f/+;-Cre;Z/AP eyes
(Fig. 2A-D,J-M), suggesting
that proper Notch1 activity is not susceptible to haploinsufficiency and that
the combination with Cre or AP expression alone does not disrupt retinal
development.
To characterize the cell-autonomous functions of Notch1, the cells in which
Cre was active were identified by monitoring AP activity in control
(Notch1f/+;-Cre;Z/AP) and Notch1
mutant (Notch1f/f;-Cre;Z/AP) eyes
(Fig. 2I-Q). Corresponding with
the early activity of -Cre in most RPCs of the embryonic peripheral
optic cup (E13.5; Fig. 2M), AP
was detected in the postnatal eyes in all cellular layers of the peripheral
retina (P15, P0; Fig. 2J-L). In
the Notch1f/f;-Cre;Z/AP eyes at E13.5, the
distribution of AP was similar to that of the control and seemed to encompass
most of the peripheral retina, including the rosettes
(Fig. 2Q), suggesting that the
rosettes are formed initially from Notch1- cells. However,
at P0 and later at P15, extensive reduction in the AP+ region was
detected in the Notch1f/f;-Cre;Z/AP retina
(Fig. 2N-P), as compared with
the size of the AP+ domain detected in the
Notch1f/+;-Cre;Z/AP
(Fig. 2J-L) eyes. Furthermore,
the rosettes at P0 seemed to contain a mixture of both AP- and
AP+ cells (Fig. 2P
inset, black arrowhead). Along with the observed reduction of AP+
cells, the size of the retina of
Notch1f/f;-Cre embryos was extensively
reduced to about 73% of that of control littermates at E15.5 (n=6 for
each genotype, P<0.001).
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-Cre;Z/AP at postnatal stages
(Fig. 2J,N,L,P, red arrowheads)
(Ashery-Padan, 2002). These
AP+ cells within the central retina were detected in the postnatal
Notch1f/f;-Cre;Z/AP eyes
(Fig. 2N, red arrowhead), thus
suggesting that Notch1 activity is redundant for the survival and normal
morphology of cells already committed to the amacrine cell fate at the time of
Notch1 inactivation.
Inactivation of Notch1 alters the expression profile of bHLH transcription factors
Notch1 activity in mammals, as in invertebrates, has been shown to inhibit
cell differentiation by upregulation of the E(Spl)-related Hes transcription
factors, which in turn repress the expression of proneural bHLH genes
(de la Pompa et al., 1997;
Hatakeyama and Kageyama, 2004;
Ohtsuka et al., 1999). Thus,
downregulation of Hes gene expression and a change in the expression profile
of proneural bHLH factors is expected to follow Notch1 receptor
inactivation.
Cre-mediated recombination of the Notch1f allele has
been shown to inactivate Notch1 in the immune system and the cerebellum
(Fig. 1A)
(Lutolf et al., 2002;
Radtke et al., 1999). To
validate the functional inactivation of Notch1 in the
Notch1f/f;-Cre retina, we monitored the
expression of Hes5 and Hes1 in the
Notch1f/f;-Cre;Z/AP and control
Notch1f+;-Cre;Z/AP retinas of E13.5
embryos, when the initial morphological abnormality becomes evident
(Fig. 2, see Fig. S1 in the
supplementary material). In the
Notch1f/+;-Cre;Z/AP embryos,
Hes5 and Hes1 expression was detected in the peripheral
optic cup overlapping with AP expression
(Fig. 3B, see Fig. S1B in the
supplementary material). In the peripheral retina of the
Notch1f/f;-Cre;Z/AP, however, Hes5 and
Hes1 expression was dramatically downregulated in the AP+ region,
while in the central (AP-) retina Hes5/1 expression was
comparable with normal (Fig.
3I, see Fig. S1D in the supplementary material). The
downregulation of Hes5/1 in the
Notch1f/f;-Cre embryos validates the
notion that -Cre-mediated deletion of the
Notch1f allele results in functional inactivation of the
Notch1 receptor in the peripheral optic cup.
|
).
The Notch1-null embryos die before the eye is formed, thus precluding the
study of Notch1-dependent bHLH expression in the retina
(Conlon et al., 1995;
Swiatek et al., 1994). We
therefore used in situ hybridization to compare the expression profile of the
proneural genes Neurod1, Ngn2, Mash1, Math5 (Atoh7 - Mouse
Genome Informatics) and Math3 (Neurod4 - Mouse Genome
Informatics) in the Notch1f/f;-Cre retinas
(Fig. 3J-N) to their expression
in control Notch1f/f retinas at E13.5
(Fig. 3A-G). In
Notch1f/f control retinas, the expression of Neurod1,
Ngn2, Mash1, Math5 and Math3
(Fig. 3C-G, respectively) was
detected in subtypes of RPCs corresponding with their intrinsic heterogeneity
(Akagi et al., 2004). In
Notch1f/f;-Cre E13.5 peripheral retina
however, increased expression of Neurod1, Ngn2, Math5 and
Math3 was detected in the distal retina
(Fig. 3J,K,M,N, respectively,
demarcated with black arrowheads). No apparent change was detected in the
expression of these genes at E12.5, prior to the onset of distinguishable
morphological phenotypes (data not shown). In contrast to previous
observations (de la Pompa et al.,
1997), Mash1 expression seemed unchanged in the
Notch1f/f;-Cre retina when compared with
the control (Fig. 3E,F
respectively). bHLH factors have been shown to cross-regulate each other's
expression (Akagi et al., 2004;
Mu et al., 2005). For example,
Mash1 expression is upregulated in Ngn2-/- and
Math3-/- retinas (Akagi
et al., 2004), thus suggesting possible inhibition of Mash1
expression by Ngn2 and Math3. It is therefore possible that upregulation of
Ngn2 and Math3 in the Notch1- optic cup inhibits Mash1
upregulation in Notch1f/f;-Cre
retinas.
Reduced proliferation and decreased proportion of ganglion and horizontal cells in the Notch1- retina
Notch activity has been linked by several studies to cell-cycle regulation
with effects that are context specific (Bao
and Cepko, 1997; Dorsky et
al., 1995; Ohnuma et al.,
2002; Scheer et al.,
2001). The consequence of Notch1 inactivation on the mitotic index
was therefore examined in the embryonic retina (E14.5) by BrdU assay. The
proportion of BrdU+ area to total nuclear area (DAPI+
area) in the optic cup was calculated (Fig.
4A-C, Materials and methods). A significant reduction (of 21.3%)
in proliferating cells was detected in the
Notch1f/f;-Cre E14.5 retina, where only
25.8% of the DAPI+ area was BrdU+, compared with the
-Cre control retina, where 32.7% of the DAPI+ area
was BrdU+ (Fig. 4C).
This suggests that due to premature differentiation of the
Notch1- cells, cell proliferation is compromised resulting
in the depletion of the RPC pool.
The involvement of Notch1 in cell survival has been demonstrated in several
cases (Lutolf et al., 2002;
Oishi et al., 2004). Hence,
the reduction in retinal size following Notch1 inactivation could also be
attributed to apoptosis. We therefore tested for programmed cell death by
TUNEL assay on sections from E14.5 and E16.5
Notch1f/f;-Cre and control peripheral
optic cup. In the control embryos, TUNEL+ cells were detected in
only 0.054% of DAPI+ area at E14.5 (s.d.=0.018%, n=4 eyes)
and 0.035% of DAPI+ area at E16.5 (s.d.=0.014%, n=3 eyes).
A significant increase (P<0.02) in the number of TUNEL+
cells was observed in the Notch1f/f;-Cre
retinas to 0.325% of DAPI+ area at E14.5 (s.d.=0.044%, n=4
eyes) and 0.28% of DAPI+ area at E16.5 (s.d.=0.1%, n=4
eyes). Thus, although a six-(at E14.5) to eightfold (at E16.5) increase in
apoptosis is observed following Notch1 inactivation in the RPCs, the
proportion of TUNEL+ cells to total retinal tissue is very low and
therefore could not solely account for the extensive reduction in retinal
tissue detected in the Notch1f/f;-Cre
retina.
|
; Austin et al.,
1995; Silva et al.,
2003). In contrast to these observations, analysis of the
phenotype of Hes1 mutant embryos revealed premature differentiation of
precursors into rod and horizontal cells
(Tomita et al., 1996). To
examine the role of Notch1 directly in the specification of RPCs to ganglion,
horizontal and photoreceptor cell fates, we characterized the expression of
markers specific to these cell types in the normal and
Notch1f/f;-Cre retina (Figs
4,
5). Brn3b is the earliest known
marker for ganglion cell precursors and is required for their terminal
differentiation and survival (Gan et al.,
1996; Gan et al.,
1999; Wang et al.,
2000). Surprisingly, at E17.5, Brn3b expression seemed to be
reduced in Notch1f/f;-Cre when compared
with the control retina (compare Fig. 4E to
4D) and a significant reduction (t-test
P<0.05) in the in the proportion of Brn3b+ to
DAPI+ area was detected peripheral retina of
Notch1f/f;-Cre E16.5 embryos: from an
average of 13.9% in the control
(Notch1f/+;-Cre; s.d.=1.7%, n=3
eyes) to an average of 8.6%
(Notch1f/f;-Cre; s.d.=0.3%, n=3
eyes). For quantitative evaluation exclusively of the
Notch1- cellular phenotype, we calculated the proportion
of Brn3b+ cells from the AP+ population in
dissociated retinas from
Notch1f/f;-Cre;Z/AP or
Notch1f/+;-Cre;Z/AP E14 age-matched
embryos (Fig. 4E-K). The
average proportion of Brn3b+;AP+ from total
AP+ in the Notch1f/+;-Cre;Z/AP
retinas was found to be 13% (Fig.
4K). By contrast, the proportion of
Brn3b+;AP+ from total AP+ was only 4.4% in
the Notch1f/f;-Cre;Z/AP retinas
(Fig. 4K). Thus, Notch1
inactivation at this stage does not seem to inhibit ganglion cell genesis;
rather, Notch1 appears to be required for their production in normal
numbers.
To detect differentiating horizontal cells, we characterized the expression
of Nf165 in the Notch1f/f;-Cre and
-Cre control embryos at E17.5, when the expression of Nf165
reaches the peripheral region of the optic cup. As early as E17.5
(Fig. 4), as well as at
postnatal stages (data not shown), Nf165+ cells seem to be
virtually excluded from the Notch1f/f;-Cre
peripheral retina. Interestingly, however, in the Hes1-/-
retina, premature differentiation into horizontal cells has been previously
reported (Tomita et al.,
1996). The different phenotypes of the Hes1 and Notch1 retina
mutants reflect the fact that Hes1 is only one of many factors that mediate
Notch1 activity (see Discussion).
Increased numbers of photoreceptor precursors following Notch1 inactivation in RPCs
We next evaluated the acquisition of photoreceptor cell fate by the
Notch1- RPCs. To identify the photoreceptor precursors in
the embryonic retina, we characterized the expression of Otx2 and Crx, members
of the otd/Otx homeobox gene family, by in situ hybridization. Otx2
is essential for photoreceptor specification, while Crx is important for their
terminal differentiation (Chen et al.,
1997; Furukawa et al.,
1997; Furukawa et al.,
1999; Nishida et al.,
2003). In the Notch1f/f E13.5 and E16.5
retinas, Otx2 (Fig. 5A,C) and
Crx (Fig. 5B,D) were weakly
detected in the outer neuroblastic layer (NBL)
(Nishida et al., 2003). In
contrast to this normal pattern of expression, as early as E13.5, both Otx2
and Crx expression were detected in the outer and inner layers of the
peripheral Notch1f/f;-Cre;Z/AP retina
(Fig. 5F-I). This suggests that
Notch1 functions to inhibit progenitors from adopting a photoreceptor fate by
repressing the expression of two key mediators of photoreceptor cell fate:
Otx2 and Crx.
We next quantified the proportion of photoreceptor precursors in the
control and Notch1f/f;-Cre embryonic
retinas by calculating the ratio of Crx+ area from the peripheral
retinal area at E16.5. In the control eyes, Crx expression is detected in
14.2% (s.d.=0.93%, n=4 eyes) of the peripheral retina. By contrast,
in the Notch1f/f;-Cre eyes,
Crx+ cells are detected in 56.6% (s.d.=9.8%, n=4 eyes) of
the distal retina (P<0.001). This fourfold increase in the
proportion of Crx+ cells in the
Notch1f/f;-Cre mice, when compared with
the control littermates at E16.5, demonstrates the depletion of the RPCs
available for the formation of other cell fates.
|
). Peak
expression is reported for Thrß2 at mid-embryogenesis (E13.5-E17.5)
(Jones et al., 2003;
Ng et al., 2001). We therefore
characterized Thrß2 expression by in situ analysis at E16.5.
Corresponding with previous reports, Thrß2 expression was restricted to
the outer nuclear layer of the
Notch1f/+;-Cre retina at E16.5
(Fig. 5E). In the
Notch1f/f;-Cre retina, however, Thrß2
expression was found to be upregulated in the outer and inner layers of the
peripheral optic cup in a pattern resembling Crx distribution identified on
adjacent sections (Fig. 5J).
The enhanced expression of Otx2, Crx and Thrß2, as well as the early
cell-cycle exit following Notch1 inactivation in the RPCs, indicated premature
differentiation of the Notch1- RPCs to photoreceptor
precursors.
To test if these precursors differentiate to mature photoreceptors
prematurely, we analyzed in the embryonic (E16.5) and early post-natal (P5)
Notch1f/f;-Cre and control retinas the
expression of several markers that are normally expressed in mature
photoreceptors. In the embryonic retina (E16.5) the expression of recoverin, a
photoreceptor-specific Ca2+-binding protein
(Haverkamp and Wassle, 2000;
Sharma et al., 2003), S-cone
opsin, Nr2e3 a rod-specific nuclear receptor important for the development of
photoreceptors (Chen et al.,
2005; Cheng et al.,
2004; Peng et al.,
2005) and rhodopsin were tested. Furthermore, the expression of
cone-arrestin (Arr3) for the detection of cones and the -subunit of rod
transducin (Gnat1) for the detection of rods were tested in P5 eyes.
Expression of none of these markers was detected in the control or
Notch1f/f;-Cre retinas (data not shown).
This suggests that the Notch1- photoreceptor precursors do
not complete their differentiation into photoreceptors prematurely.
The Notch1- photoreceptor precursors differentiate primarily into cone-photoreceptors
The cellular phenotype acquired by the Notch1-
precursors was characterized after all retinal cell types had been born (P15)
(Young, 1985), by detecting
markers specific to the retinal cell types using antibody labeling or in situ
hybridization (Fig. 6). The
analysis was conducted on
Notchf/+;-Cre;Z/AP controls and
Notch1f/f;-Cre;Z/AP mice in order to
distinguish between Notch1- (AP+) and
Notch1+ (AP-) cells
(Fig. 1). AP enzymatic activity
was detected either on adjacent sections
(Fig. 6) or when the antibody
reaction protocol permitted, by double-immunolabeling with antibodies specific
to the different retinal cell types (see Fig. S2 in the supplementary
material).
First, we analyzed the expression of recoverin, which is expressed in
differentiated photoreceptors (Haverkamp
and Wassle, 2000; Sharma et
al., 2003). In the
Notchf/+;-Cre;Z/AP control retina, AP
expression was detected in all of the cellular layers of the peripheral retina
(Fig. 6A) while recoverin
expression was detected exclusively in the photoreceptor layer
(Fig. 6B). In the
Notch1f/f;-Cre;Z/AP peripheral retina,
however, recoverin expression (Fig.
6E) was detected in almost all of the AP+ cells
(Fig. 6D). Quantitative
analysis of the proportion of recoverin from AP expressing cells in the
peripheral retina of the control and the
Notch1f/f;-Cre;Z/AP mice was performed
(see Material and methods, Fig.
6M). In the control peripheral retina (AP+), recoverin
was detected in 56.7% of the nuclear (DAPI+) area
(Fig. 6M). By contrast, in the
AP+ regions of the
Notch1f/f;-Cre;Z/AP retinas recoverin was
detected in 96.7% of the DAPI+ area
(Fig. 6M). Thus, consistent
with the increased number of photoreceptor precursors observed during
embryogenesis following Notch1 inactivation, a dramatic increase in the
proportion of differentiated photoreceptors is observed in the postnatal
Notch1- retina. In agreement with the detection of
recoverin in most of the AP+ cells of the peripheral retina of the
Notch1f/f;-Cre;Z/AP mice, co-expression of
AP with syntaxin (amacrine cells), PKC (bipolar cells) and cyclin D3
(Müller glia cells) was not observed in the peripheral retina of
Notch1f/f;-Cre;Z/AP mice, while these
cell-specific markers were co-localized with AP-expression in the
Notchf/+;-Cre;Z/AP controls (see Fig. S2
in the supplementary material).
To further characterize the photoreceptor cell types produced from the
Notch1- RPCs, we identified cone-photoreceptors by
labeling sections with cone-specific peanut agglutinin (PNA) conjugated to
fluorescein (Blanks and Johnson,
1983). Rods were identified by immunofluorescent analysis for
detection of rhodopsin in the control and the
Notch1f/f;-Cre;Z/AP eyes
(Fig. 6C,F). AP activity was
monitored on adjacent sections (Fig.
6A,D). In the control
Notchf/+;-Cre;Z/AP retina, rhodopsin
expression and PNA labeling were confined to the outer segments of the
photoreceptor (Fig. 6C) and
only a few cells were labeled with PNA, corresponding to the low number of
cones present in the mouse retina (Young,
1985). By contrast, in the
Notch1f/f;-Cre;Z/AP peripheral retina, a
dramatic increase in the number of PNA+ cells was detected in
AP+ regions (identified on adjacent section
Fig. 6D), while
rhodopsin+ cells were identified mostly in AP- regions
(Fig. 6F). To further validate
cone-fate acquisition, the expression of cone arrestin (Arr3) for the
detection of cones and of the -subunit of rod transducin (Gnat1) for
the detection of rods was characterized in the control and
Notch1f/f;-Cre;Z/AP retina by in situ
hybridization (Fig. 6G-L). The
proportion of the retinal area expressing each of these markers in the
AP+ regions was calculated (measurements were conducted on the area
that was AP+ detected on adjacent sections,
Fig. 6M). Consistent with the
normal differentiation pattern observed in the
Notchf/+;-Cre;Z/AP control retina, Arr3
(cones) was detected in few cells of the outer nuclear layer while Gnat1
(rods) was detected in most of the outer nuclear layer
(Fig. 6H,I). In the peripheral
retina (AP+) of the control mice Arr3 was detected in 2% of the
DAPI+ area, while Gnat1 was detected in 66.7% of the
DAPI+ area (Fig.
6M). However, in the
Notch1f/f;-Cre;Z/AP peripheral retina
(AP+; demarcated with broken white line
Fig. 6J-K), Arr3 was detected
in 89.1% of the DAPI+ area, while Gnat1 was detected in 10.1% of
the DAPI+ area (Fig.
6M). This demonstrates that the Notch1- RPCs
differentiate predominantly into cone-photoreceptors.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Austin et al., 1995;
Bernardos et al., 2005;
Dorsky et al., 1997;
Furukawa et al., 2000;
Perron and Harris, 2000;
Rapaport and Dorsky, 1998;
Silva et al., 2003). In this
case, it follows that premature loss of Notch1 activity in the RPCs of the
peripheral retina during early embryogenesis (as in the
Notch1f/f;-Cre mice) would result in the
excessive differentiation of Notch1- RPCs into cell types
normally born during the corresponding early stages of retinogenesis:
ganglion, cone, horizontal and amacrine cells
(Fig. 7)
(Young, 1985). Consistent with
its proposed role as an inhibitor of cell differentiation, we indeed observed
that the selective targeted deletion of Notch1 in early RPCs leads to their
premature terminal exit from the cell cycle, and their differentiation and
accumulation within rosette-like structures. Surprisingly, however, the
premature loss of Notch1 in RPCs leads to the acquisition of primarily the
cone-photoreceptor cell fate. Intriguingly, these Notch1-
RPC-derived cone precursors appear to be specified at the expense of the other
early cell types.
|
|
;
Oishi et al., 2004).
Furthermore, the aberrant retinal morphology is expected to result in cell
death. The findings presented in this study, however, seem to rule out
selective cell death as the primary explanation for reduced retinal size and
the higher number of cone precursors in the
Notch1f/f;-Cre retina. Although we
observed enhanced apoptosis at E14.5 and E16.5 in the
Notch1f/f;-Cre when compared with control
littermates, this cell death was not extensive (covering only 0.32% at E14.5
or 0.28% at E16.5 of Notch1f/f;-Cre retina
tissue). This suggests that cell death does not account for the massive
reduction in retina size or for the fourfold increase in Crx+ cells
in the Notch1f/f;-Cre retina. Although the
above findings do not exclude selective cell death of a specific low-abundance
progenitor cell type, it appears that the reduced retinal tissue and the
abnormal cellular composition is primarily due to the precocious specification
of Notch1- progenitors to cone precursors, which results
in the depletion of the RPCs destined to other cell fates.
Notch1 functions to inhibit cone-photoreceptor cell fate specification during early retinal neurogenesis
The current model for retinal development suggests that retinal precursors
consist of mixed subsets of progenitors with distinct competence states that
are subject to changes over time, presumably driven by the shifting presence
of external and internal cues (Livesey and
Cepko, 2001; Watanabe and
Raff, 1990). Cell-birth studies reveal that during early
retinogenesis, the ganglion, cone, horizontal, amacrine and rod cells are born
from the pool of RPCs (between E12 and E13)
(Seidman, 1961;
Young, 1985). Thus, one might
expect that premature differentiation during early retinogenesis will result
in early differentiation of the progenitors into these early cell types. Our
finding that the Notch1- RPCs acquire predominantly the
cone fate raises two non-mutually exclusive models for explaining the
mechanisms controlling cell-type diversification in the retina: first, these
findings may suggest that there is an underlying bias toward the
cone-photoreceptor fate of early RPCs. The forced early differentiation
imposed by the inactivation of Notch1 in the
Notch1f/f;-Cre merely exposes this
dominant bias to the cone-photoreceptor fate. However, this possibility seems
less likely as the cone precursors are never the predominant cell type
(Young, 1985). Another
explanation for the observed
Notch1f/f;-Cre retinal phenotype is that
Notch1 maintains the multipotency of RPCs by selectively suppressing
acquisition of the cone-photoreceptor fate. Consistently, premature loss of
Notch1 activity in early RPCs leads to excessive production of cone
precursors, thereby depleting the pool of RPCs available for formation of the
other retinal cell types.
Involvement of Notch1 pathway in acquisition of retinal cell fates
Several intrinsic and extrinsic factors appear to regulate the onset of
cell differentiation and cell-type specification by influencing the expression
of bHLH proneural factors. In Hes1-null mutant mice, rod
photoreceptor and horizontal cells were reported to appear prematurely during
retinogenesis (Tomita et al.,
1996), suggesting that loss of Hes1 alone leads to a late-onset
phenotype compared with that observed for the Notch1-deficient retina. These
phenotypic differences are consistent with the notion that Notch1 acts through
several pathways to control retinal neurogenesis, in addition to a late-acting
Hes1-dependent pathway. For example, in the Hes1-null mutant retina,
persistent expression of other Hes genes, such as Hes5, could potentially lead
to a rescue of earlier aspects of neurogenesis. In this regard, the earlier
and more dramatic phenotype observed in our Notch1-deficient retinal model may
be at least partially explained by the loss of expression of both Hes1 and
Hes5.
Another key regulator of eye development known to be essential for the
multipotency of RPCs is Pax6 transcription factor. The inactivation of Pax6 in
RPCs resulted in downregulation of Math5, Ngn2 and Mash1, while Neurod1
expression seemed unchanged. This change in expression profile was associated
with the RPCs acquiring the amacrine cell fate
(Marquardt et al., 2001). The
different expression profile of the proneural bHLH factors in the Pax6 when
compared with Notch1 mutants is consistent with the difference in eventual
cell fate acquired by the cells; amacrine fate by Pax6-
RPCs and cone-photoreceptor fate by the Notch1- RPCs.
Recently, the secreted factor GDF11 has been suggested to control cell-type
specification of progenitor cells in the developing retina by regulation of
Math5, Mash1 and Neurod1 expression (Kim
et al., 2005). Together, these studies suggest that several
factors and pathways intersect in terms of regulating the expression of the
bHLH transcription factor profile in the progenitor cells. In future studies,
it would be intriguing to discover the interaction between these different
pathways at the onset of differentiation and in mediating the specification of
multiple retinal cell types.
The development of photoreceptors has been studied extensively as a model
for understanding neuronal specification and differentiation
(Cepko et al., 1996).
Photoreceptor differentiation has been shown to occur in two phases. Cones are
one of the first cells to appear (between E11 and E13), together with the
other early-born cell types: ganglion, horizontal and amacrine. The second
phase of photoreceptor differentiation is marked by the production of rods
that peaks around birth (Sidman,
1961; Young,
1985). The transcription factors Otx2 and Crx are the main factors
currently known to regulate the early events of photoreceptor specification
and differentiation (Chen et al.,
1997; Furukawa et al.,
1997; Nishida et al.,
2003). Using the Cre/loxP approach, Otx2 was found to be
essential for the formation of photoreceptor precursors, while its target,
Crx, is required for their normal differentiation
(Chen et al., 1997;
Furukawa et al., 1997;
Nishida et al., 2003). The
expression and function of Otx2 in the eye, however, seems not to be
restricted to photoreceptor precursors and thus cannot be the only mediator of
Crx expression (Martinez-Morales et al.,
2001).
The results presented here suggest that Notch1 is functionally upstream of
Otx2 and Crx, and it acts to inhibit the expression of both
genes in early RPCs (Fig. 7).
Future studies will have to address the identity of the mediators that
regulate Otx2 and Crx expression downstream of Notch1
activation. Following Notch1 inactivation in the RPCs, we observed
down-regulation of Hes5 and Hes1 and enhanced expression of
a number of proneural genes, such as Neurod1, Math5, Math3 and
Ngn2. This radically altered combination of bHLH factors expressed in
the Notch1- cells could therefore underlie the observed
change in cell fate toward cone-photoreceptor precursors
(Fig. 7). Furthermore, in
previous studies using gain- and loss-of-function approaches, the
rod-photoreceptor cell fate has been shown to be affected by combinations of
several bHLH factors (Akagi et al.,
2004; Ma et al.,
2004; Morrow et al.,
1999; Pennesi et al.,
2003; Yan and Wang,
1998). It would therefore be interesting in future studies to
evaluate the regulation of Otx2 and Crx by the combination of bHLH proneural
factors and to assess more carefully the acquisition of the cone cell fate in
bHLH mouse mutants.
Although many of the photoreceptor precursors are generated during
embryogenesis, the differentiation into functional photoreceptors is completed
mostly at postnatal stages (Morrow et al.,
1998b). This long interval between photoreceptor birth and onset
of visual pigment expression has been observed in many different species
(Adler et al., 2001;
Knight and Raymond, 1990;
Morrow et al., 1998a). We
observed that the cone precursors originating from Notch1-deficient RPCs seem
to obey the normal cone-photoreceptor developmental program. Similar to normal
cone precursors, the expression of recoverin, S-opsin, Arr3 and PNA labeling
was detected only after birth. Together, the presented findings demonstrate an
important role for the Notch1 pathway in actively preventing RPCs from
choosing the cone-photoreceptor fate, a role that appears, however, to be
independent of the later temporal process that leads to the maturation of
committed cone-photoreceptor precursors.
|
ACKNOWLEDGMENTS |
|---|
-Cre mice, and Drs Corrinne Lobe
and Andras Nagy for the Z/AP reporter line. For the in situ probes,
we thank Drs Ryoichiro Kageyama, Thomas A. Reh, Takahisa Furukawa, Jichao Chen
and Jeremy Nathans. Antibodies were generous gifts from Karl W. Koch
(recoverin) and Nadean Brown (Hes1). We are grateful for the assistance of
Leonid Mittleman for confocal analysis and for Noa Davis-Silberman's comments
on the manuscript. R.A.-P.'s research is supported by the Israeli Academy of
Sciences, Israeli Ministry of Health, the German Israeli Foundation, Recanati
Foundation, ADAMS Center for Brain Studies. O.Y. is supported by a Fred Shaul
Scholarship. M.L.A.'s research is supported by NIH Grants, Macular
Foundation/Ronald McDonald House Charities and the Massachusetts Lions Eye
Research Fund. |
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/7/1367/DC1
* These authors contributed equally to this work 
|
REFERENCES |
|---|
|
TOP
SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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