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RESEARCH ARTICLE
127
Development 138, 127-138 (2011) doi:10.1242/dev.053637
© 2011. Published by The Company of Biologists Ltd
Roles for Dicer1 in the patterning and differentiation of the
optic cup neuroepithelium
Noa Davis, Eyal Mor and Ruth Ashery-Padan*
SUMMARY
The embryonic ocular neuroepithilium generates a myriad of cell types, including the neuroretina, the pigmented epithelium, the
ciliary and iris epithelia, and the iris smooth muscles. As in other regions of the developing nervous system, the generation of
these various cell types requires a coordinated sequence of patterning, specification and differentiation events. We investigated
the roles of microRNAs (miRNAs) in the development of optic cup (OC)-derived structures. We inactivated Dicer1, a key mediator
of miRNA biosynthesis, within the OC in overlapping yet distinct spatiotemporal patterns. Ablation of Dicer1 in the inner layer of
the OC resulted in patterning alteration, particularly at the most distal margins. Following loss of Dicer1, this region generated a
cryptic population of cells with a mixed phenotype of neuronal and ciliary body (CB) progenitors. Notably, inactivation of Dicer1
in the retinal progenitors further resulted in abrogated neurogenesis, with prolongation of ganglion cell birth and arrested
differentiation of other neuronal subtypes, including amacrine and photoreceptor cells. These alterations were accompanied by
changes in the expression of Notch and Hedgehog signaling components, indicating the sensitivity of the pathways to miRNA
activity. Moreover, this study revealed the requirement of miRNAs for morphogenesis of the iris and for the regulation of CB cell
type proliferation and differentiation. Together, analysis of the three genetic models revealed novel, stage-dependent roles for
miRNAs in the development of the ocular sub-organs, which are all essential for normal vision.
INTRODUCTION
Normal vision depends on differentiation of the optic
neuroepithelium into neuronal and non-neuronal ocular sub-organs:
the neuroretina, retinal pigmented epithelium (RPE), the ciliary
body (CB) and the iris. The neuroretina is populated by six classes
of neuron and Müller glia that are organized in three cell layers.
The outer nuclear layer (ONL) contains the cone and rod
photoreceptors, whereas the inner nuclear layer (INL) is populated
by the bipolar cells, horizontal cells, amacrine cells (ACs) and
Müller glia. Finally, residing in the ganglion cell layer (GCL) are
the displaced ACs and ganglion cells (GCs). Adjacent to the
photoreceptor layer is the RPE, a cuboidal epithelial layer of
pigmented cells essential for the maintenance and function of the
photoreceptors (Strauss, 2005). Anterior to the retina and RPE are
the ocular structures of the anterior segment of the eye: the CB and
the iris. The CB is connected to the lens through zonular fibers and
is composed of two epithelial layers that are tightly associated: a
posterior non-pigmented layer and anterior pigmented layer. A
primary role for the CB is secretion of the aqueous humor, and of
components of the vitreous and inner limiting membranes. The CB
is therefore pivotal to the establishment and maintenance of ocular
pressure and to the survival of the GCs (Gould et al., 2004; Halfter
et al., 2008; Halfter et al., 2005). The iris is composed of two layers
of pigmented epithelium, muscles and stroma (for a review, see
Davis-Silberman and Ashery-Padan, 2008). The iris controls the
Sackler Faculty of Medicine, Department of Human Molecular Genetics and
Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel.
*Author for correspondence ([email protected])
Accepted 20 October 2010
amount of light entering the eye and is involved in the circulation
of the aqueous humor (Cvekl and Tamm, 2004; Davis-Silberman
and Ashery-Padan, 2008).
Normal development of these structures provides a model
system with which to investigate early patterning, gradual
specification and subsequent differentiation into the mature cell
types (Yang, 2004; Esteve and Bovolenta, 2006). FGFs secreted
from the surface ectoderm and TGF from the surrounding ocular
mesenchyme specify the RPE and neuroretina domains in the outer
and inner layers of the optic cup (OC). The interface between the
RPE and the neuroretina is the region that gives rise to the anterior
structures. The inner layer of the OC contains the retinal progenitor
cells (RPCs), which differentiate into the retinal cell types in a
defined spatiotemporal order. GCs, cone photoreceptors and
horizontal cells are formed first, followed by ACs and rod
photoreceptors, with bipolar and Müller cells appearing last
(Young, 1985).
The generation of neuronal and non-neuronal cell types from the
OC is spatially and temporally regulated by intrinsic and extrinsic
cues. Wnt and BMP signaling promote the non-neuronal fates,
whereas Shh and Notch1 are involved in neurogenesis (Jadhav et
al., 2006a; Jadhav et al., 2006b; Liu et al., 2007; Yaron et al., 2006;
Yu et al., 2006; Zhao et al., 2002). Differentiation of the RPE and
retina follows a center to periphery pattern, progressing towards the
OC periphery and arresting at the distal-most tips, where the
progenitors of the CB and iris reside (Davis-Silberman and AsheryPadan, 2008).
Formation of the retina and anterior structures of the iris and CB
depends on tight spatiotemporal regulation of gene expression.
MicroRNAs (miRNAs) are small non-coding RNAs, which have
emerged as key regulators of developmental events across phyla
(Lee and Ambros, 2001; Pasquinelli et al., 2000; Reinhart et al.,
2000). It has been suggested that most vertebrate genes are
DEVELOPMENT
KEY WORDS: Dicer1, MicroRNAs, Iris, Ciliary body, Optic cup, Retinogenesis, Mouse
RESEARCH ARTICLE
regulated by miRNAs (Friedman et al., 2009b). miRNAs control
gene expression post-transcriptionally by regulating either the
stability or translation efficiency, or both, of different mRNAs. A
single miRNA might directly alter the expression of hundreds of
proteins to a mild extent, and indirectly affect the expression of
thousands more (Baek et al., 2008; Selbach et al., 2008). Although
the effect on the levels of individual proteins might be subtle, the
summed effect is significant. Indeed, particular miRNAs have been
linked to the formation of specific organs, demonstrating their
crucial role during development (Maatouk and Harfe, 2006).
Primary miRNA transcripts are processed by two RNaseIII
ribonucleases, Drosha and Dicer1 (Grishok et al., 2001; Giraldez
et al., 2006), to form an ~22-bp miRNA duplex that is incorporated
into the RNA-induced silencing complex (RISC). Although Dicer1
mutant mice die on embryonic day (E) 7.5 (Bernstein et al., 2003),
a conditional inactivation approach enabled a study of its role in
various organs, exposing the role of miRNAs in multiple
developmental processes (e.g. Harfe et al., 2005; Harris et al.,
2006).
Dozens of miRNAs have been detected in the mouse eye, some
showing dynamic expression patterns during ocular development
(Hackler et al., 2010; Xu et al., 2007). Targeting of Dicer1 in
Xenopus embryos revealed its importance for cell-cycle exit,
survival and differentiation timing of retinal cells (Decembrini et
al., 2008). In mammals, conditional mutagenesis of Dicer1 was
established using Chx10-Cre, which is active in a mosaic pattern in
the retina but not in the non-neuronal progenitors of the CB and
iris. The phenotype of Dicer1loxP/loxP;Chx10-Cre mutants included
reduced survival of photoreceptors and improper lamination of the
retina (Damiani et al., 2008). In a recent study, Dicer1 activity was
explored in the inner layer of the OC by employing the a-Cre
transgene (Georgi and Reh, 2010). That study revealed important
roles for Dicer1 in the generation and survival of all retinal cell
types. These intriguing data encouraged us to explore miRNA
involvement in the patterning of the OC, in the generation of the
derived non-neuronal structures and in the signaling pathways that
might mediate the pleiotropic functions of miRNAs in the retina
and the adjacent ocular sub-organs.
MATERIALS AND METHODS
Mice
The transgenic mouse lines Tyrp2-Cre, Z/AP, -Cre, Pou4f3-Cre and
Dicer1loxP/loxP were established as described previously (Davis et al., 2009;
Lobe et al., 1999; Marquardt et al., 2001; Sage et al., 2006; Harfe et al.,
2005) and Z/AP mouse lines were established as described previously
(Davis et al., 2009; Lobe et al., 1999; Marquardt et al., 2001; Sage et al.,
2006). Genotyping was performed by PCR using the primers listed in Table
S1 in the supplementary material. All procedures involving animals
followed NIH guidelines and were approved by the Animal Care and Use
Committee of Tel Aviv University.
The day on which the copulatory plug was observed was defined as
E0.5, and the day of birth was referred to as postnatal day 1 (P1). For
pulse-chase experiments, pregnant females were injected with BrdU (140
g/g body weight) and embryos or eyes were harvested at the specified
ages. Specimens were fixed in 4% paraformaldehyde at 4oC, dehydrated in
a graded ethanol series, and embedded in paraffin wax. Alternatively, fixed
tissues were cryoprotected in 30% sucrose and embedded in OCT. For
histological staining, 10 m paraffin sections were stained with
Hematoxylin and Eosin (H&E) according to a standard protocol. Alkaline
phosphate (AP) staining was performed on either 10 m paraffin or 14 m
frozen sections as described previously (Lobe et al., 1999). For
immunofluorescence analysis, either paraffin or frozen sections were
stained as described by Marquardt et al. (Marquardt et al., 2001).
Antibodies used were hAP (1:100, Santa Cruz), Pax6 (1:400, Convance),
Development 138 (1)
Cx43 (1:1000, Sigma), Cav3 (1:100, Santa Cruz), PCNA (1:100, Dako),
Ki67 (1:100, Dako), CcnD1 (1:250, Thermo Scientific), CcnD3 (1:100,
Santa Cruz), p57kip2 (1:50, Santa Cruz), Pou4f2 (1:100, Santa Cruz), Rho
(1:1000, generous gift from M. Applebury, Harvard Medical School,
Boston, MA, USA), NF165 (1:500, Developmental Studies Hybridoma
Bank), PKC (1:1200, Santa Cruz), syntaxin 1 (1:500, Sigma), Crlbp
(1:1000, generous gift from J. C. Saari, University of Washington, Seattle,
WA, USA), Sox2 (1:500, Chemicon), PH3 (1:500, Santa Cruz), Isl1 (1:100,
Developmental Studies Hybridoma Bank), BrdU (1:100, Chemicon),
Chx10 (1:1000, Exalpha), Mitf1 (1:1000, generous gift from K. Bharti,
NINDS, Bethesda, MD, USA), cCas3 (1:300, Cell Signaling), PNA (1:200,
Vector Laboratories). For in situ hybridization, frozen sections were stained
with DIG- or POD-labeled probes as reported by Yaron et al. (Yaron et al.,
2006). Probes used were Otx1 and Bmp4 (generous gift from P. Bovolenta,
Instituto Cajal, Madrid, Spain), Notch1 (Schroder and Gossler, 2002), Hes5
(Ohtsuka et al., 1999), Neurod1 (Lee et al., 1995), Ptc1 (generous gift from
C. L. Cepko, Harvard Medical School, Boston, MA), Crx (Furukawa et al.,
1997), Foxn4 (generous gift from M. Xiang, Robert Wood Johnson
Medical School, Piscataway, NJ, USA) and Gli1 (Furimsky and Wallace,
2006). Each experiment included at least three different animals from each
genotype. For in situ hybridization of miR-204 we used LNA-probe
(Exiqon), following the manufacturer’s instructions but with a minor
modification as described by Xu et al. (Xu et al., 2007).
Measurements and morphometric analyses
To obtain position-matched sections, eyes were embedded parallel to the
optic nerve and transverse serial sections were collected. For quantification
of GCs in the neuroblastic layer (NBL), Pou4f2+ or Isl1+ cells in the Ki67+
region were counted and normalized to area. At least four sections per
animal were averaged. Recombinant regions were defined using the hAP
reporter gene. To assess the thickness of the GCL, we measured its area
and divided it by overall retinal width.
RESULTS
Roles for miRNAs in development of the iris and
the CB
The neuroretina and most of the iris and CB cell types originate
from adjacent progenitors of the OC. To explore the potential role
of miRNAs in the formation of these structures, we employed three
Cre-transgenic lines, each with unique but partly overlapping
activity in OC progenitors. The pattern of Cre activity was
monitored by AP expression from the Z/AP reporter transgene (Fig.
1A,F,K; Fig. 2A,F) (Marquardt et al., 2001; Davis et al., 2009;
Davis-Silberman et al., 2005; Lobe et al., 1999). The
recombination activity of Tyrp2-Cre is initiated on around E11 in
the ocular pigmented cell types (Davis et al., 2009). At late
embryogenesis, it is expressed in the presumptive pigmented
epithelia of the CB and the iris (Fig. 1A,F), and then in the muscles
and stroma of the iris (Davis et al., 2009). The -Cre line is active
within the inner layer of the OC from E10.5, including in the
progenitors of the neuroretina, the non-pigmented CB and the iris
(Marquardt et al., 2001). Later, -Cre activity overlaps with that of
Tyrp2-Cre in the iris pigmented epithelia (IPE) and muscles (Fig.
1A,F,K) (Davis-Silberman et al., 2005). Finally, Pou4f3-Cre is
instrumental for conditional mutagenesis in postnatal development
of the anterior ocular structures, owing to its ectopic expression in
the non-pigmented CB and the posterior IPE starting from P1 (Fig.
2A,F). At later stages, it is expressed in iris muscle as well (not
shown). In contrast to the endogenous expression of Pou4f3, we
did not detect Cre activity in the GCL.
Mutant mice from all genotypes displayed a variety of
abnormalities. Dicer1loxP/loxP;Tyrp2-Cre adult mice showed poor
survival and small body size, probably owing to Tyrp2-Cre
expression in the developing forebrain and dorsal root ganglia
(Davis et al., 2009). Similarly, most Dicer1loxP/loxP;Pou4f3-Cre
DEVELOPMENT
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Roles for microRNAs in the optic cup
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129
mutants died soon after birth, possibly due to a malformation of the
upper jaw that impedes normal suckling (Friedman et al., 2009a).
Mutant eyes of both genotypes, but not of Dicer1loxP/loxP;-Cre
mice, showed severe microphthalmia and lacked the vitreous cavity
(Fig. 1G,L; data not shown). We next examined morphogenesis of
the anterior structures of the mutants. First, we compared the
formation of the CB in Dicer1loxP/loxP;Tyrp2-Cre and
Dicer1loxP/loxP;-Cre mutants. The recombination pattern of the
Tyrp2-Cre transgene included the pigmented CB, whereas the Cre transgene was active in the non-pigmented layer (Fig. 1A).
Nevertheless, the CB of both mutants was flattened and lacked the
characteristic undulating processes (Fig. 1B,G,H,L). To verify the
identity of the hypoplastic CB, we determined the expression of
Pax6 and Cx43. After birth, Pax6 is normally expressed in retinal,
iris and CB cell types, but not in the RPE, whereas Cx43 is highly
expressed in the CB (Coffey et al., 2002; Walther and Gruss, 1991)
(Fig. 1D,E). Both Cx43 and Pax6 were detected in the
Dicer1loxP/loxP;Tyrp2-Cre and Dicer1loxP/loxP;-Cre CBs (Fig.
1I,J,N,O), suggesting that the initial specification occurred but later
morphogenesis was abrogated. These findings reveal an important
role for miRNA in CB development.
In contrast to the comparable phenotype of the mutant CBs of
these two mouse lines, the iris reacted differently. Both Cre lines
were active within the IPE (Fig. 1A); however, the Tyrp2-Cre
transgene was also expressed in the iris stroma (Davis et al., 2009).
This divergence resulted in a dramatic change in the
morphogenesis of the mutant iris. On P8, the iris is fully formed,
including the IPE, muscles and stroma (Fig. 1C). However,
histological analysis of the Dicer1loxP/loxP;Tyrp2-Cre mutants
revealed that most of the iris tissues were missing, with only a thin
strand of Pax6+ cells remaining (Fig. 1H,I). In the
Dicer1loxP/loxP;-Cre mutant the iris did form, including the
pigmented epithelia, stroma and muscles (Fig. 1M). The main
defect exhibited by the P8 Dicer1loxP/loxP;-Cre iris was
detachment of the two epithelial layers (Fig. 1M). Taken together,
we concluded that miRNAs in the stroma are pivotal for iris
formation, whereas their existence in the iris epithelia mainly
contributes to cell-cell adhesion.
In both the -Cre- and Tyrp2-Cre-mediated deletions of Dicer1,
miRNA excision takes place at the progenitor stage, which
potentially prevents the analysis of their late role in CB and iris
differentiation. We therefore used the Pou4f3-Cre line, expressed
postnatally in the anterior structures (Fig. 2A,F). Similar to
Dicer1loxP/loxP;-Cre line, the IPE was detached (Fig. 2G), although
the sphincter muscle, marked with the specific antibody caveolin3
(Cav3) (Kogo et al., 2006), exhibited a distinctive overgrowth
phenotype (Fig. 2G,H, compare with control Fig. 2B,C). The CB
was hyperplastic and disorganized, and the two epithelial layers
were detached (Fig. 2I,J, compare with control Fig. 2D,E).
Hyperplasia might result from increased proliferation; indeed,
Ki67+ cells were detected in the detached non-pigmented CB (Fig.
2J), which was in contrast to their complete absence in control
siblings (Fig. 2E). These findings suggest that miRNAs are
required postnatally to restrain growth of the CB and sphincter
muscle, and that their requirement for epithelial adherence might
occur after birth.
DEVELOPMENT
Fig. 1. miRNA are required for the formation of the iris and the CB in mouse. (A)Scheme of Cre-mediated recombination monitored by
detection of human alkaline phosphatase (hAP) in the Tyrp2-Cre;Z/AP (F) and -Cre;Z/AP (K) lines at E18.5. (B,C,G,H,L,M) H&E staining of whole
eyes (B,G,L) or irises (C,H,M) of control (B,C), Dicer1loxP/loxP;Tyrp2-Cre (G,H) and Dicer1loxP/loxP;-Cre (L,M) mice. (D,E,I,J,N,O) Immunofluorescence
staining with Pax6 (D,I,N) or Cx43 (E,J,O) of control (D,E), Dicer1loxP/loxP;Tyrp2-Cre (I,J) and Dicer1loxP/loxP;-Cre (N,O) eyes. Inset in D shows higher
magnification of the iris. CB, ciliary body; IPE, iris pigmented epithelium; Ir, iris; IS, iris stroma; Le, lens; NPCB, non-pigmented ciliary body; Nr,
neuroretina; PCB, pigmented ciliary body; Sp, sphincter; VC, vitreous cavity. Scale bars: 500m (B,G,L); 100m (C-F,H-K,M-O).
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Development 138 (1)
Fig. 2. miRNA are required postnatally to restrain the growth of the CB and iris. (A)Scheme of Cre mediated-recombination monitored by
detection of the hAP reporter gene in Pou4f3;Z/AP pups at P1 (F). (B,D,G,I) H&E staining of adult iris (B,G) and CB (D,I) in control (B,D) and
Dicer1loxP/loxP;Pou4f3-Cre (G,I) eyes. (C,E,H,J) Protein distribution of Cav3 (C,H) or Ki67 (E,J) in control (C,E) or Dicer1loxP/loxP;Pou4f3-Cre (H,J) mice.
Inset in J is a higher magnification of the boxed area. CB, Ciliary body; IPE, iris pigmented epithelium; Le, lens; Nr, neuroretina; NPCB, nonpigmented ciliary body; PCB, pigmented ciliary body; Sp, sphincter. Scale bars: 100m (B,F,G); 50m (C-E,H-J).
retina (Fig. 3B-D; data not shown) (Calera et al., 2006; Lee et al.,
2001; Martinez-Morales et al., 2001), and Otx1 and Bmp4 have
been related to CB development (Larsen et al., 2009; Zhao et al.,
2002). In Dicer1loxP/loxP;-Cre mutants, the expression domains of
Cx43, Otx1 and Gas1 were markedly decreased compared with in
control littermates (Fig. 3G,H; data not shown). Moreover, Bmp4
expression, normally seen on the apical side of the retinal tips, was
downregulated in Dicer1loxP/loxP;-Cre E15.5 embryos and P2 pups
(Fig. 3D,E,I,J). These data suggest that Dicer1 ablation hampers
the development of the CB and that miRNAs are involved in its
prenatal specification or differentiation.
The Dicer1-deficient distal retina is populated by a
mixture of neuronal and non-neuronal progenitors
We further used the -Cre line to study miRNA function within the
neuroretinal compartment of the OC. In contrast to the normal wide
expression pattern of -Cre, the expression region was diminished
Fig. 3. Molecular alterations in the CB of Dicer1loxP/loxP;-Cre mutants. (A,F)In situ hybridization of miR-204 on control (A) and
Dicer1loxP/loxP;-Cre (F) eyes. (B,G)Immunofluorescence staining with Cx43 and (C-E,H-J) in situ hybridization of Otx1 and Bmp4 in (B-E) control and
(G-J) Dicer1loxP/loxP;-Cre eyes. Arrowheads in B-E and G-J indicate the presumptive CB. Insets in B,E,G,J are higher magnifications of the iris-CB
domain. The eyes in C,E,J are pigmented. Co, cornea; Le, lens; NPCB, non-pigmented ciliary body; Nr neuroretina; PCB, pigmented ciliary body.
Scale bars: 100m (B-D,G-I); 50m (A,E,F,J).
DEVELOPMENT
To delve into the molecular etiology associated with abrogated
development of the CB, we focused on Dicer1loxP/loxP;-Cre
mutants. In order to determine the extent of Dicer ablation, we
characterized the expression of miR-204, which is known to be
expressed in the postnatal CB (Karali et al., 2007; Ryan et al.,
2006). At around P7, miR-204 is highly expressed in the pigmented
and the non-pigmented layers of the CB (Fig. 3A). In the
Dicer1loxP/loxP;-Cre mutants, miR-204 expression was absent from
the non-pigmented CB layer, corresponding with -Cre activity
(Fig. 3F). This demonstrates that compensation by wild-type cells
is not prevalent in the CB of Dicer1loxP/loxP;-Cre mutants.
On P7, the Dicer1loxP/loxP;-Cre CB already appeared flattened
and misshapen (Fig. 3F). We therefore studied Dicer1loxP/loxP;-Cre
at younger ages. Although CB morphogenesis occurs mostly
postnatally, differential gene expression distinguishes it from the
adjacent neuroretina during embryogenesis. Cx43, Gas1, Otx1 and
Bmp4 are expressed in the progenitors of the CB but not of the
Roles for microRNAs in the optic cup
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131
in the Dicer1loxP/loxP;-Cre mutants (see Fig. S1A,B,E,F in the
supplementary material). This decrease correlated with the
extensive apoptosis detected in the mutants from E14.5 onwards
(Decembrini et al., 2008; Georgi and Reh, 2010) (see also Fig.
S1G,H in the supplementary material).
Histological staining showed that unlike the differentiated,
stratified structure of the normal retina (Fig. 4A), the mutant retina
was hypocellular and atypically consisted of columnar progenitorlike cells (Fig. 4F). We characterized the phenotype of the retinal
rudiment using markers for neuroretina and CB progenitors (Fig.
4). By P8, most of the retinal cells have already exited the cell
cycle and downregulated the expression of progenitor markers (Fig.
4B,C). By contrast, Dicer1–/– retinal cells were positive for a
variety of progenitor markers, including PCNA, Ki67 (Fig. 4G,H),
Pax6, Ccnd1, p27kip1 (also known as Cdkn1b) and Sox2 (not
shown). Intriguingly, we observed upregulation of Ccnd3 and
p57kip2 (Cdkn1c) (Fig. 4I,J), which are either absent or rarely
expressed in the normal RPCs, respectively (Fig. 4D,E) (Dyer and
Cepko, 2000). To delve into this phenomenon, we characterized the
expression patterns of p57kip2 and Ccnd3 within the OC. On
E18.5, p57kip2 was detected in selected ACs (Dyer and Cepko,
2000), whereas Ccnd3 was absent from the retina. Both markers
were weakly expressed in the CB primordium on E18.5 (Fig. 4K)
and were upregulated after birth (Fig. 4L). In the Dicer1loxP/loxP;Cre retina, expression of p57kip2 and Ccnd3 was low on E18.5 and
dramatically upregulated after birth (Fig. 4O,P), thus suggesting an
acquisition of CB progenitor characteristics by the Dicer1–/– retina.
Moreover, the expression of Pax6 was detected in most of the distal
retinas of mice of the Dicer1loxP/loxP;-Cre line on P1 (Fig. 4Q),
similar to its high expression in the CB and iris epithelia (Fig. 4M,
arrowhead). Worth mentioning is that this was not the case in either
Dicer1loxP/loxP;Tyrp2-Cre or Dicer1loxP/loxP;Pou4f3-Cre eyes. We
thus concluded that this aberrant population originates from either
the non-pigmented CB or the retina itself, both of which are wild
type in Dicer1loxP/loxP;Tyrp2-Cre mice. Moreover, the emergence of
this population is expected to occur prenatally, prior to the onset of
Pou4f3-Cre activity.
Despite the expression of markers indicative of non-neuronal
identity, the Dicer1-deficient retina also contained cells expressing
the GC markers Pou4f2 (previously known as Brn3b; Fig. 4R) and
beta-III-Tubulin (Tubb3; data not shown). Other retinal neurons did
not differentiate in the Dicer1loxP/loxP;-Cre mutants, including
photoreceptors (rodopsin and PNA; see Fig. S2B,G in the
supplementary material), horizontal cells (NF165) (see Fig. S2C,H
DEVELOPMENT
Fig. 4. The Dicer1-deficient distal OC is populated by a mixture of neuronal and non-neuronal progenitors. (A,F)H&E staining and
(B-E,G-R) antibody staining for PCNA (B,G), Ki67 (C,H), Ccnd3 (D,I,K,L,O,P), p57kip2 (E,J,K,L,O,P), Pax6 (M,Q) and Pou4f2 (N,R) in (A-E,K-N) control
mice and (F-J,O-R) Dicer1loxP/loxP;-Cre mutants. Insets in B-E show the expression pattern of these markers in the CB at P8. The intense staining
seen in E is of the zonular fibers. Arrowheads in K-M,O-Q indicate the presumptive CB. CB, Ciliary body; Le, lens; Nr, neuroretina. Scale bars:
50m.
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Development 138 (1)
in the supplementary material), bipolar cells (PKC) (see Fig.
S2C,H in the supplementary material), ACs (syntaxin 1) (see Fig.
S2D,I in the supplementary material) and Müller glia (Crlbp) (see
Fig. S2E,J in the supplementary material). The analysis of the
neuronal subtypes formed in Dicer1-null retina corresponds with a
recent study on the retinal phenotype of Dicer1loxP/loxP;-Cre
mutants (Georgi and Reh, 2010). Importantly, our results reveal
that the postnatal retinal rudiment of Dicer1loxP/loxP;-Cre mice
contains a mixture of distal progenitors, as well as GCs. These
findings implicate miRNA in the establishment of the border
between the neuronal and non-neuronal progenitors of the OC.
Furthermore, as the Dicer1loxP/loxP;Tyrp2-Cre mice did not exhibit
the aberrant progenitor domain, we concluded that miRNAs in the
progenitors of the iris and the pigmented CB are required for
normal differentiation of the end sub-organs, whereas their function
in the inner OC is in the partitioning of neuronal and non-neuronal
progenitors.
We next characterized the patterning of the Dicer-null OC nonneuronal and neuronal compartments. On E15.5, neuroretinal
progenitors divided extensively and expressed Sox2, Ccnd1 and
Ki67. These proteins were expressed at low levels in the CB
progenitors, which divide slowly at this stage (Fig. 5A-C).
Additional evidence for the border between the neuronal and nonneuronal compartments was the positioning of the GCL, which on
E15.5 was abutting the CB-progenitor pool (Fig. 5). In
Dicer1loxP/loxP;-Cre retinas, the GCL and the CB anlagen were
spatially separated by aberrant progenitor cells, which expressed
high levels of the progenitor cell markers Sox2, Ccnd1 and Ki67,
thus creating a ciliary margin-like region (Fig. 5E,F,G,Q).
Furthermore, an elevated number of phospho-H3+ cells was seen
in this mutant region, indicative of increased mitotic activity (Fig.
5H).
A major player in the establishment of retinal identity is Chx10
(also known as Vsx2), whereas Mitf1 is a key regulator of
pigmentation (Graw, 1996; Bharti et al., 2006). Mutual repression
between Chx10 and Mitf1 defines the neuroretinal and pigmented
progenitor domains within the OC (Horsford et al., 2005; Rowan
et al., 2004). We thus monitored the expression of Chx10 and Mitf1
in Dicer1loxP/loxP;-Cre embryos. On around E12.5 and E14.5 in
control mice, Chx10 was expressed in most of the inner OC layer,
abutting the marginal Mitf1-expressing cells (Fig. 5I,J). At later
stages, an intermediate zone, expressing low levels of Chx10 and
Mitf1, became evident between the neuronal (Chx10 high; Fig.
5K,L) and non-neuronal (Mitf1 high) domains. In the
Dicer1loxP/loxP;-Cre embryos, however, Chx10 levels were
elevated distally (Fig. 5M,N) and the intermediate zone was absent
(Fig. 5O,P). In agreement with the mutual-repression assumption,
the expression of Mitf1 was reduced in the distal cells (Fig. 5MO). This alteration in Chx10 and Mitf1 expression could contribute
to the severe hypoplasia of the CB and to the formation of the
cryptic progenitors identified in the postnatal retinas of
Dicer1loxP/loxP;-Cre mice.
DEVELOPMENT
Fig. 5. Dicer1 ablation leads to the formation of an ectopic progenitor population within the ciliary margins. (A-P)Antibody staining for
Sox2 (A,E), Ccnd1 (B,F) Ki67 (C,G), PH3 (D,H), Chx10 (I-P) and Mitf (I-P) in (A-D,I-L) control mice and (E-H,M-P) Dicer1loxP/loxP;-Cre mutants.
(Q)Schematic illustration of the retina/CB-iris presumptive domains in control (Top) and Dicer1loxP/loxP;-Cre (Bottom) eyes. Insets in A-H are higher
magnifications of the iris-CB domain; L and P are higher magnifications of the boxed regions in K and O, respectively. Arrowheads in A-H indicate
the edge of the GCL. CB, Ciliary body; GCL, ganglion cell layer; Le, lens; Nr, neuroretina. Scale bars: 100m (A-H); 50m (I-P).
Roles for microRNAs in the optic cup
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133
Fig. 6. Dicer1-null retina displays differentiation failure of
amacrine cells and photoreceptors. (A,D,G-L) In situ hybridization
for Foxn4 (A,D), Neurod1 (G,J) and Crx1 (H,I,K,L), and (B,C,E,F)
Antibody staining for Ptf1a and Ap2a (B,E), and bHLH5 and syntaxin 1
(C,F), in (A-C,G-I) control and (D-F,J-L) Dicer1loxP/loxP;-Cre eyes. GCL,
Ganglion cell layer; INL, inner neuroblastic layer; Le, lens; NBL,
neuroblastic layer; RPE, retinal pigmented epithelium. Scale bars:
50m.
Differentiation failure of amacrine and
photoreceptor cells in the Dicer1-deficient retina
The postnatal Dicer1loxP/loxP;-Cre retina was composed of
progenitor cells intermingling with GCs (Fig. 4). This, however,
did not exclude the possibility that other cell types are born but fail
to fully differentiate. We therefore investigated the specification of
the amacrine and photoreceptor lineages.
Foxn4 is expressed in cycling precursors of ACs and horizontal
cells (Fig. 6A) (Li et al., 2004). Ptf1a expression rose in early
postmitotic amacrine precursors, similar to expression of basic
helix-loop-helix 5 (bHLH5), which is also found in differentiating
ACs after they settle in the prospective INL (Fig. 6B,C) (Fujitani
et al., 2006; Feng et al., 2006). Ap2a (also known as Tcfap2a) and
syntaxin 1 were detected in ACs migrating to and settling in the
INL (Fig. 6B,C). In the E18.5 Dicer1loxP/loxP;-Cre retina,
expression of the early specification gene Foxn4 and Ptf1a was
maintained (Fig. 6D,E). By contrast, the precursor factors Ap2a
and bHLH5 showed altered distribution (Fig. 6E,F), whereas
syntaxin 1 was completely abolished (Fig. 6F). These results are in
agreement with data showing that the inner plexiform layer was not
formed in the mutants (see Fig. S2D in the supplementary
material), and imply that miRNAs are dispensable for specification
but essential for differentiation of the AC types.
miRNAs are required to inhibit GC production
throughout the OC
Extensive production of GCs was observed throughout the Dicer1deficient retinas. We therefore examined the dynamics of GC
formation in Dicer1loxP/loxP;-Cre mutants. GCs are the earliest
retinal cell type, with the last cell born on around E14.5 (Young,
1985). The GC precursors migrate to and populate the GCL (Fig.
7A,L). In Dicer1loxP/loxP;-Cre embryos, however, a considerable
number of Pou4f2+ cells populated the NBL on E15.5 and E18.5
(Fig. 7E,P). Double staining with the cell-cycle marker Ki67
demonstrated an 841% elevation in the number of Pou4f2+ cells in
the NBL on E18.5 (s.d.152%, n5, P>0.001) in comparison to in
the control retinas (s.d.28.4%, n3). Accordingly, the GCL was
46% thicker in the mutants (s.d.14%, n5, P<0.001) than in the
controls (s.d.5%, n3).
To confirm the identity of these cells as GCs and to rule out a
specific upregulation of Pou4f2, we performed the same analysis
with Isl1. Normally, Isl1 is expressed in a distinct, yet overlapping
population of GCs, as well as in differentiating ACs and bipolar
cells (Mu et al., 2008). On E18.5, Isl1+ cells are mostly confined
to the GCL and to the presumptive INL (Fig. 7B). In the mutants,
we observed a higher proportion of Isl1+ cells in the NBL (631%,
s.d.126%, n4, P<0.005) than in the NBL of controls (s.d.31%,
n2), supporting the notion that the mislocated Pou4f2+ cells are
indeed GCs (Fig. 5F).
The presence of GCs in the NBL could be interpreted as an
uncoupling of proliferation and differentiation. To test this, we
performed double staining with cell-cycle markers (i.e. Ki67 and
PCNA) and Pou4f2 at various developmental stages (E15.5, E17.5,
P1 and P8). We found that soon after the birth of the GCs, there is
a short temporal overlap between the decay of cell-cycle markers
and the onset of Pou4f2 expression. In control retinas, this
phenomenon was seen until E15.5 but, in the mutants, it extended
to perinatal days (not shown). This finding implied that the
uncoupling of proliferation and differentiation is not impeded in the
absence of Dicer1, but that the timeframe of the birth of GCs might
be prolonged. We therefore performed a pulse-chase experiment in
which embryos were injected with BrdU on E17.5 and harvested
on either P1 or P8. In P1 control embryos, BrdU+ cells were
detected in the presumptive INL and ONL (Fig. 7C). On P8,
BrdU+ cells populated the INL and ONL, with a few BrdU+ cells
in the GCL, marking displaced ACs (Fig. 7D). No colocalization
DEVELOPMENT
We then investigated the photoreceptor fate. We studied the
expression of Crx1, a key factor in the formation of photoreceptors
and one of their earliest markers (Fig. 6H,I) (Furukawa et al.,
1997). In E15.5 Dicer1loxP/loxP;-Cre retinas, Crx1 transcripts were
correctly detected in the scleral aspect of the embryonic retina (Fig.
6K). On P2, however, Crx1 expression was lost from the Dicer1deficient retina (Fig. 6L). Neurod1 is a bHLH transcription factor
known to be involved in AC differentiation and photoreceptor
survival (Morrow et al., 1999). On E18.5, it is highly expressed
along the outer aspect of the developing retina, with punctate
staining within the NBL (Fig. 6G) (Morrow et al., 1999). In the
Dicer1loxP/loxP;-Cre retinas, Neurod1 expression was lost from the
outer layer, with only a few cells maintaining expression in the
NBL (Fig. 6J). This finding might be related to the loss of both
ACs and photoreceptors in Dicer1loxP/loxP;-Cre retinas. Taken
together, this analysis corresponds with a recent study on Dicer1
deficiency in RPCs (Georgi and Reh, 2010), and is suggestive of
normal specification followed by a complete failure to differentiate
to the AC and photoreceptor lineages.
134
RESEARCH ARTICLE
Development 138 (1)
of BrdU and Pou4f2 was observed in the P1 or P8 control mice. In
Dicer1loxP/loxP;-Cre retinas, however, BrdU+;Pou4f2+ cells were
prevalent on P1 (Fig. 7G), but not on P8 (Fig. 7H), suggesting that
some GCs were anachronistically born on E17.5, but failed to
survive until P8.
To explore molecular alterations related to the aforedescribed
phenomena, we examined the notch and hedgehog (Hh) pathways,
which are implicated in neurogenesis and GC production (Jadhav
et al., 2006a; Jadhav et al., 2006b; Riesenberg et al., 2009; Yaron
et al., 2006; Furimsky and Wallace, 2006; Sakagami et al., 2009;
Wang et al., 2005). Notch1 and its downstream target Hes5 are
normally expressed in the neuronal, but not in the non-neuronal,
progenitors of the OC (Fig. 7I,J) (Bao and Cepko, 1997; Kageyama
and Ohtsuka, 1999; Yaron et al., 2006). In Dicer1loxP/loxP;-Cre
retinas, however, marked downregulation of Notch1 and Hes5
transcript was detected on around E17.5 (Fig. 7M,N). Examination
of Dicer1loxP/loxP;-Cre retinas demonstrated that the ganglion
phenotype is already evident on E15.5 (Fig. 7L,P). Nonetheless,
Notch1 expression was normal at this stage, whereas Hes5
appeared elevated when compared with littermate controls (Fig.
7Q,R,U,V). This might reflect a diminution of precursors rather
than a direct alteration in Hes5 expression. This analysis therefore
did not support involvement of the Notch pathway in mediating the
elevation in GC production.
DEVELOPMENT
Fig. 7. Dicer1 ablation leads to
expanded production of
ganglion cells and alterations in
the Notch1 and Hh pathways.
(A-H,L,P) Antibody staining for
Pou4f2 (A,C,D,E,G,H,L,P), Isl1 (B,F),
hAP reporter gene (B,F) and BrdU
(C,D,G,H), and (I-K,M-O,Q-X) in
situ hybridization for Notch1
(I,M,Q,U), Hes5 (J,N,R,V) Gli1
(K,O,S,W) and Ptc1 (T,X) in (A-D,IL,Q-T) control and (E-H,M-P,U-X)
Dicer1loxP/loxP;-Cre eyes. Insets in
C,D,G,H are higher magnifications
of the boxed area in the
corresponding images. The eyes in
I-K and in T are pigmented. GCL,
Ganglion cell layer; INL, inner
nuclear layer; Le, lens; NBL,
neuroblastic layer; Nr, neuroretina;
ONL, outer nuclear layer; RPE,
retinal pigmented epithelium. Scale
bars: 50m (A,B,E,F); 100m
(C,D,G-X).
To monitor Hh activity in Dicer1loxP/loxP;-Cre retinas, we
analyzed the expression of Gli1, a transcriptional mediator and
downstream target of the Hh pathway (Parisi and Lin, 1998). In
E17.5 control retinas, Gli1 displayed dual expression: high in the
NBL and low in the GCL (Fig. 7K) (Dakubo et al., 2008). In
Dicer1loxP/loxP;-Cre retinas, however, only a low level of Gli1
transcript was detected (Fig. 7O). Decreased expression was also
evident on E15.5, a finding that coincides temporally with the
formation of extra GCs in the mutants (Fig. 7S,W). To verify the
inhibition of Hh signaling at this stage, we looked at the expression
of Patched1 (Ptc1), a co-receptor of Smo and a downstream target
of the pathway, and found a similar downregulation on E15.5 (Fig.
7T,X). Considering the delay in miRNAs degradation described in
Dicer1 conditional lens mutation (Li and Piatigorsky, 2009) and
that the changes in GC distribution were initiated after E12.5 (data
not shown), we conclude that the inhibition of Hh signaling is a
relatively early result of Dicer1 ablation and correlates with the
misregulation of GC production.
DISCUSSION
This study describes the roles of Dicer in the developing retina and
anterior structures of the eye cup. Dicer1 was found to be required
for the partitioning of neuronal and non-neuronal progenitors in the
distal OC, for differentiation of most retinal cell types and for
regulation of timing of production and number of GCs. The
changes caused by ablation of Dicer1 were associated with
perturbations in the expression of Notch- and Hh-signalingresponsive genes. Furthermore, the study revealed a requirement
for miRNAs in the adhesion properties of epithelial cells of the iris
and CB, and in restricting growth of the CB and iris cell types at
postnatal stages.
Tissue-specific roles for miRNA in iris
development
The iris forms from two embryonic origins: the OC, which gives
rise to the muscles and the epithelia; and the periocular
mesenchyme, which contributes to the stroma (Davis-Silberman
and Ashery-Padan, 2008). Here, we identified miRNA functions
within the different iridial tissues during embryonic and postnatal
development. The phenotype of Dicer1loxP/loxP;-Cre mutants
suggests that miRNAs in the inner OC are dispensable for the
formation of the IPE and muscles, but act at later stages in the
adherence of the epithelial layers (Fig. 1M). Interestingly,
detachment of epithelial cells following Dicer1 loss has been
described in lung epithelium and kidney cell types (Harris et al.,
2006; Harvey et al., 2008; Ho et al., 2008). This, together with our
findings, implicates a general role for miRNA in the regulation of
cell-adherence-related molecules.
Despite the overlapping expression of the -Cre and Tyrp2-Cre
transgenes within the iris, the latter Cre line was uniquely active
within the iris stroma. The severity of the iridial phenotype of
Dicer1loxP/loxP;Tyrp2-Cre mutants indicates the importance of
Dicer1 activity in the stroma for morphogenesis of the adjacent iris
layers. The formation of the stroma includes migration of
mesenchymal cells into the anterior eye, adherence to the iris
scaffold, proliferation and differentiation (Cvekl and Tamm, 2004;
Smith, 2002). All of these biological processes can potentially
involve miRNA regulation of gene expression.
Finally, the use of the late-acting Pou4f3-Cre transgene enabled
us to examine the role of miRNA in the late differentiation of the
iris, precluding the early patterning defects of the Dicer1loxP/loxP;Cre. Surprisingly, late ablation of Dicer1 in the anterior structures
RESEARCH ARTICLE
135
led to a persistence of cycling cells in the mature CB and to a
marked overgrowth of the sphincter muscle (Fig. 2H,J). Owing to
the mosaic pattern of recombination mediated by the Pou4f3-Cre
transgene, the phenotypes might at least partly reflect a non-cellautonomous response of Dicer+ cells to their mutant neighbors.
This finding suggests that miRNA might act to restrain the size of
the vision accessorial structures, a function needed for the
coordinated growth of the eye.
CB formation requires Dicer1 function
The first event occurring during morphogenesis of the murine
ciliary processes is a proliferative surge of the pigmented
epithelium (Napier and Kidson, 2005). After a slight delay, the
non-pigmented epithelium responds by proliferating and extending
inward towards the lens. This coordinated development entails
close contact of the two epithelial layers. Indeed, targeting the gap
junction or cell-adhesion molecules Cx43 and nectin-1/nectin-3
(also known as Pvrl1/Pvrl3), localized at the junctions between the
pigmented and non-pigmented layers, disrupts formation of the CB
(Calera et al., 2006; Inagaki et al., 2005). Here, we show that
exclusive ablation of Dicer1 from either the non-pigmented
(Dicer1loxP/loxP;-Cre) or pigmented (Dicer1loxP/loxP;Tyrp2-Cre)
layers seriously hampers the development of both epithelia,
emphasizing the vital and reciprocal role of the two layers in the
formation of each other (Fig. 1I,J,N,O). Furthermore, targeting
Dicer1 activity on postnatal days using the Pou4f3-Cre line
demonstrated that this role is mainly played at the progenitor stage,
whereas at postnatal stages miRNAs act primarily to restrain CB
growth (Fig. 2I,J). To date, the only miRNA localized to the CB
epithelium has been miR-204 (Ryan et al., 2006) (Fig. 3A). A
recent study demonstrated reduced expression of miR-204 in a
NCI60 tumor cell line panel, suggesting an inhibitory function on
cell proliferation (Wang et al., 2010). In the same study, miR-204
was implicated in maintenance of the epithelial barrier in
pigmented epithelium. The loss of miR-204 might mediate the
increase in cell divisions, as well as the epithelial detachments,
seen in Dicer1loxP/loxP;Pou4f3-Cre and Dicer1loxP/loxP;-Cre mice.
miRNAs are required for late patterning of the
distal OC
Several pieces of evidence, mostly from amphibians and birds,
suggest that partitioning of the neuroretina and CB domains is
labile up to at least the late stages of development (Mitashov,
2001; Fischer and Reh, 2001; Moshiri et al., 2004; Spence et al.,
2004). In mammals, it is not clear when the borders between the
neuronal and non-neuronal compartments of the OC are
irreversibly defined. An indication of two distinct progenitor pools
is evident in the mouse at around mid-gestation (Davis et al.,
2008). Cell-cycle-related factors are differentially expressed by the
progenitors of both the CB and the retina, with a decreasing
gradient from retina to CB (Fig. 5A-C) (Fischer and Reh, 2000;
Liu et al., 2007). Our data demonstrate that cells located at the
distal tip of Dicer1loxP/loxP;-Cre embryos ectopically express high
levels of progenitor markers (Fig. 5E-G). The elevated expression
of Chx10 in these cells (Fig. 5O,P) and the decreased size of the
CB domain (Fig. 3G,H) suggest that this ectopic upregulation
results from an undermining of the non-neuronal identity of the
distal cells. Close to birth, however, this cryptic population
upregulates the expression of CB-progenitor genes, with a timing
that matches their normal onset within the CB (e.g. Ccnd3, p57
and Pax6; Fig. 4O-Q). At the same time, the expression of some
neuronal factors [Neurod1 and Crx1 (Fig. 6J,K,L); Notch1 and
DEVELOPMENT
Roles for microRNAs in the optic cup
RESEARCH ARTICLE
Hes5 (Fig. 7M,N)] decreases. It is possible that these molecular
alterations merely result from ablation of certain miRNAs;
however, when considering the embryonic distal origin of these
cells, together with the upregulation of Chx10, it is tempting to
speculate that the mutant cells possess a mixed CB/retinal identity.
This interpretation proposes a novel role for miRNAs in the
maintenance of the neuronal/non-neuronal border of the OC.
Notably, despite the broad area of -Cre expression (see Fig.
S1A,E in the supplementary material), only cells located at the
retina-CB junction acquired the cryptic phenotype. This is further
supported by the fact that ablation of Dicer1 by Chx10-Cre, which
is mostly active in patches of the proximal retina, does not result
in the described changes (Damiani et al., 2008).
miRNA involvement in retinal differentiation
Several studies have correlated miRNAs and differentiation
potential. In Caenorhabditis elegans, post-transcriptional regulation
of the heterochronic gene lin-14 by lin-4 mediates temporal pattern
formation (Wightman et al., 1993), and the 21-nucleotide let-7
RNA regulates developmental timing (Reinhart et al., 2000).
Knockout of either Dicer1 or DGCR8, an RNA-binding protein
involved in miRNA processing, results in differentiation failure of
embryonic stem (ES) cells (Kanellopoulou et al., 2005; Wang et al.,
2007). Finally, inactivation of Dicer1 in the Xenopus retina
increases the number of cycling cells and causes a delay in cell
birth (Decembrini et al., 2008). The latter findings correspond with
the persistence of progenitors within the postnatal Dicer1loxP/loxP;Cre retina (this study) (Georgi and Reh, 2010) and with the nonpigmented CB in Dicer1loxP/loxP;Pou4f3-Cre mice. It is worth
mentioning that there are studies (Ahmad et al., 2000; Tropepe et
al., 2000) suggesting that there is a reservoir of retinal stem cells
within the CB. Those cells, however, were derived from the
pigmented layer of the CB rather than the non-pigmented one and
thus, might not be related to the cryptic progenitors identified here
following Dicer1 inactivation.
miRNAs might safeguard against signals that could otherwise
simultaneously activate alternative differentiation programs within
the same cell lineage (Hornstein and Shomron, 2006); thus, the
observed role of miRNAs in retina/CB partitioning could reflect a
more general miRNA-based mechanism underlying the
differentiation of progenitors, along with controlled diminution of
the self-renewal program. Indeed, this idea is compatible with
previous studies addressing the roles of miRNAs in a variety of
developmental contexts. For example, miR-203 promotes
epidermal differentiation by restricting proliferative potential and
inducing cell-cycle exit (Yi et al., 2008). Similarly, miR-430
represses maternally contributed mRNA, thus promoting transition
to the zygotic program (Giraldez et al., 2006). Finally, mutually
exclusive cross-regulation of the miR-290-295 cluster and let-7 is
involved in the transition of ES cells to differentiated tissues
(Melton et al., 2010).
The regulation of proliferation versus differentiation by miRNA
might be intertwined with known regulatory mechanisms, such as
Notch and Hh signaling. Recent studies on the functions of Notchsignaling genes support a role for this pathway in maintaining the
progenitor pool and in inhibiting GC and photoreceptor lineages
(Jadhav et al., 2006b; Yaron et al., 2006; Riesenberg et al., 2009).
Dicer loss resulted in a complex, stage-dependent perturbation of
the Notch-pathway genes: Hes5 expression was elevated on E15.5
but lost on E17.5. Moreover, whereas Notch activity normally
inhibits the expression of most proneural genes (Jadhav et al.,
2006b; Yaron et al., 2006), in the Dicer1-deficient retina, Neurod1
Development 138 (1)
expression was lost and Math3 was not altered on E17.5, despite
the loss of Hes5 and Notch1 at this stage (Fig. 6K; N.D. and R.A.P., unpublished observations). These finding reflect the complex
effects of miRNAs on the Notch components and suggest that,
either directly or indirectly, miRNAs play a role in regulating the
proneural genes, in particular Neurod1.
In mammals, the conditional inactivation of Hh signaling in
RPCs leads to a bias toward a retinal ganglion fate, resulting in an
increase in the number of GCs (Sakagami et al., 2009; Wang et al.,
2001). Furthermore, it has been shown that retroviral-mediated
overexpression of Shh results in reduced GC proportions in vivo
and in vitro (Zhang and Yang, 2001). These findings support the
possibility that downregulation of the Shh targets Gli1 and Ptc1
might contribute to the GC phenotype of Dicer1–/– retina. The
challenge for future studies will be to decipher the gene networks
regulated by specific miRNA families that mediate their pleiotropic
functions in retinogenesis and development of the sub-organs of the
eye.
Acknowledgements
We are grateful to Eran Hornstein for the Dicer1loxP/loxP mice and for comments
on the manuscript, to Karen B. Avraham for the Dicer1loxP/loxP;Pou4f3-Cre mice
and comments on the manuscript, and to Paola Bovolenta, Constance L.
Cepko and Valerie A. Wallace for in-situ probes. R.A.-P.’s research is supported
by the Israel Science Foundation 610/10, the Israel Ministry of Science 36494,
the Ziegler Foundation and the Binational Science Foundation.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.053637/-/DC1
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