Progress in Retinal and Eye Research xxx (2012) 1e26
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Progress in Retinal and Eye Research
journal homepage: www.elsevier.com/locate/prer
Pax6: A multi-level regulator of ocular developmentq
Ohad Shaham a, Yotam Menuchin a, Chen Farhy a, Ruth Ashery-Padan a, b, *
a
b
Sackler Faculty of Medicine, Department of Human Molecular Genetics and Biochemistry, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel
Sagol School of Neuroscience, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxx
Eye development has been a paradigm for the study of organogenesis, from the demonstration of lens
induction through epithelial tissue morphogenesis, to neuronal specification and differentiation. The
transcription factor Pax6 has been shown to play a key role in each of these processes. Pax6 is required
for initiation of developmental pathways, patterning of epithelial tissues, activation of tissue-specific
genes and interaction with other regulatory pathways. Herein we examine the data accumulated over
the last few decades from extensive analyses of biochemical modules and genetic manipulation of the
Pax6 gene. Specifically, we describe the regulation of Pax6’s expression pattern, the protein’s DNAbinding properties, and its specific roles and mechanisms of action at all stages of lens and retinal
development. Pax6 functions at multiple levels to integrate extracellular information and execute cellintrinsic differentiation programs that culminate in the specification and differentiation of a distinct
ocular lineage.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Pax6
Lens
Retina
Eye development
Contents
1.
2.
3.
The Pax6 genedhistorical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Regulation and products of the Pax6 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Regulation of the complex expression profile of Pax6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Structure and transcriptional activity of the Pax6 proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The roles of Pax6 in specification and morphogenesis of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Specification of OV progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Pax6 is one of several eye-field genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.
Roles for EFTFs in the partitioning of the single eye field and early patterning of the OV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.
Extrinsic cues pattern the OV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
The multiple functions of Pax6 in the development of OC derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.
The development of the OC neuroepithelium to the ocular lineages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2.
Roles for Pax6 in the differentiation of OC progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3.
Pax6 in RPC cell-cycle regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Specification of the ocular surface ectoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1.
Pax6 determines the lens pre-placodal region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2.
Extrinsic cues in lens-fate determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: bHLH, basic helix-loop-helix; BMPs, bone morphogenetic proteins; BrdU, bromodeoxyuridine; CB, ciliary body; CNS, central nervous system; E#,
embryonic day #, days post-conception; ECM, extracellular matrix; EE, ectodermal enhancer; EFTF, eye field transcription factor; FGF, fibroblast growth factor; HH,
Hamburger and Hamilton; HTH, helix-turn-helix; LE, lens ectoderm; LFC, lens fiber cell; LP, lens placode; MAPK, mitogen-activated protein kinase; OC, optic cup; OV, optic
vesicle; P#, post natal day #; PAX, paired box; PE, pigmented epithelium; PPR, pre-placodal region; PST, proline serine threonine; RPC, retinal progenitor cell; RPE, retinal
pigmented epithelium; SE, surface ectoderm; SELEX, systematic evolution of ligands by exponential enrichment; TGFb, transforming growth factor b; YAC, yeast artificial
chromosome.
q Percentage of work contributed by each author in the production of the manuscript is as follows: Ohad Shaham contributed to sections 1, 3.3, 3.4 and Fig. 5. Yotam
Menuchin contributed section 2 and Fig. 1. Chen Farhy contributed to section 3.2 and Figs. 3 and 4. Ruth Ashery-Padan incorporated the different parts, wrote other sections
and prepared Fig. 2.
* Corresponding author. Sackler Faculty of Medicine, Department of Human Molecular Genetics and Biochemistry, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.
Tel.: þ972 36409331; fax: þ972 36405834.
E-mail addresses: ruthash@post.tau.ac.il, asherypadan@gmail.com (R. Ashery-Padan).
1350-9462/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.preteyeres.2012.04.002
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
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O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
4.
3.3.3.
Pax6 is required in a stage-dependent manner in the transition of PPR to lens placode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.4.
Morphogenesis of the lens placode and lens pit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.5.
A diploid level of Pax6 is required for detachment of the lens vesicle from the surface ectoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Roles of Pax6 in the transition from lens progenitors to differentiated cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.1.
Lens fiber differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.2.
Pax6 is required for differentiation of secondary lens fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.3.
Pax6 down-regulation is required at the final stages of lens differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.
Roles of Pax6 in corneal development and homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. The Pax6 genedhistorical perspective
Long before the discovery of the Pax6 gene and its role in
mammals, spontaneously established Drosophila mutant lines in
which eyes completely failed to develop were described and
termed eyeless (ey). The eyeless phenotype served as a genetic
marker and was mapped to chromosome 4 of Drosophila melanogaster (Sturtevant, 1951). More than 10 years later, and independently of eyeless, the semi-dominant and homozygous lethal
“Small Eye” (Sey) mutation was discovered in mice (Roberts, 1967).
Observations of Sey/Sey homozygous mouse embryos showed that
the optic vesicle (OV) extends laterally during early gestation, but
the head surface ectoderm (SE) fails to develop into a lens, aborting
eye morphogenesis (Hogan et al., 1986). In heterozygous (Sey-/þ)
mice, the lens did develop, but it was smaller and often remained
connected to the cornea (Hogan et al., 1986).
In humans, a semi-dominant condition termed ’congenital
aniridia’ (lack of iris) was described long before modern genetic
analyses became available (Rush, 1926). Aniridia is a recessive lethal
disease, which in the semi-dominant heterozygous state involves
a plethora of ocular abnormalities: iris and ciliary body (CB)
hypoplasia, cataract and dislocation of the lens, corneal opacity,
foveal dysplasia, glaucoma and additional pathological phenotypes
(Lee et al., 2008).
Based on gene mapping, evolutionary conservation and phenotypic similarities, it was proposed that mouse Sey and human
aniridia result from mutations in orthologous genes (Glaser et al.,
1990; van der Meer-de Jong et al., 1990). The human aniridia gene
was isolated by positional cloning and was found to contain a paired
box (Ton et al., 1991). The paired box encodes a DNA-binding
domain that was initially identified in Drosophila segmentation
and subsequently discovered in a family of pairedbox-containing
mammalian genes given the name PAX (Kessel and Gruss, 1990;
Treisman et al., 1991; Walther et al., 1991). As a member of the
PAX family, Pax6 was shown to be expressed in the murine central
nervous system (CNS), eye, olfactory system and pancreas (Walther
and Gruss, 1991). It was subsequently demonstrated that the mouse
Sey results from mutations in the Pax6 gene (Hill et al., 1991). In the
following decades, hundreds of causative mutations of aniridia
were reported within the PAX6 locus (The Human PAX6 mutation
Database see http://lsdb.hgu.mrc.ac.uk) (Brown et al., 1998; Kokotas
and Petersen, 2010; Tzoulaki et al., 2005).
In parallel, homologues of mammalian Pax6 were cloned in
zebrafish and quail (Krauss et al., 1991; Martin et al., 1992). Astoundingly, conservation of Pax6 far exceeds the mammalian or even
vertebrate context. Using sequence homology of the murine Pax6 gene,
Quiring et al. (1994) were able to identify a paired- and homeoboxcontaining gene within the eyeless locus of D. melanogaster, uncovering
the causative mutations of the eyeless phenotype inside this gene’s
coding region. Conservation of Pax6 is not limited to the DNA level.
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Ectopic expression of human Pax6 in Drosophila and Xenopus embryos
leads to the formation of displaceddyet apparently functional eye
structures (Chow et al., 1999; Halder et al., 1995; Viczian et al., 2009;
Zuber et al., 2003). This indicates that there is a highly conserved
genetic pathway triggered by Pax6 in combination with other eye
transcription factors which is able to confer eye-forming competence
to certain embryonic tissues. What remains a mystery is what aspects
in Pax6’s activity are conserved among the different species (Kozmik,
2005). Another related question is how a seemingly conserved
protein, such as Pax6, regulates the diversity of cell lineages that
populate the vertebrate eye (Arendt et al., 2009). Resolving these
questions will contribute to our understanding of the molecular
mechanisms regulating cell diversification and organogenesis, and of
how the regulatory networks evolved from the most primitive metazoan eyes to several remarkably different eye structures, including the
compound eyes of insects and the camera eyes of vertebrates.
2. Regulation and products of the Pax6 gene
2.1. Regulation of the complex expression profile of Pax6
In addition to its role in eye development, Pax6 is pivotal for
normal development of the CNS, olfactory system and pancreas,
and plays a role in adult neurogenesis (reviewed in Hanson and Van
Heyningen, 1995; Osumi et al., 2008; Simpson and Price, 2002). In
each of these developmental contexts, Pax6 displays a highly
complex spatiotemporal expression pattern with a varied dosage of
gene expression. The dynamic and complex expression pattern of
Pax6, which will be discussed in detail in the following sections, can
be attributed to regulatory DNA sequences that function in a coordinated, sometimes overlapping manner to tightly and robustly
regulate gene expression.
The mammalian Pax6 gene encompasses 16 exons spanning
roughly 28 kb: 14 exons are numbered 0e13 and the other two are
termed a and 5a (Fig. 1A, black rectangles) (Glaser et al., 1992;
Kammandel et al., 1999; Kim and Lauderdale, 2006). Several
different transcripts are synthesized at the Pax6 locus, either due to
selection of different promoters or through post-transcriptional
alternative splicing.
Two of the main transcription start sitesdP0 and P1 (Plaza et al.,
1995b; Xu and Saunders, 1997)dencode two different 13-exon long
transcripts that are translated into an identical polypeptide, as
translation of Pax6 begins at the first ATG on exon 4 (Walther and
Gruss, 1991). However P0- and P1-derived transcripts are differentially regulated during embryonic development as evidenced by
distinct spatial and temporal expression patterns of each alternative transcript in the eye and brain (Anderson et al., 2002; Xu et al.,
1999b). The biological significance of P0- and P1-derived transcripts is not yet understood. Nevertheless, it might be that the
existence of two separate promoters for the same protein allows for
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
(2012), doi:10.1016/j.preteyeres.2012.04.002
O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
3
Fig. 1. Gene, transcript and protein structure of Pax6. (A) Genomic structure of the mouse Pax6 gene (not to scale). The coding exons are colored, the non-coding exons are in black.
The regulatory elements are gray rhomboids and are designated with lowercase letters (see Table 1). Transcription start sites are marked with arrows. (B) The structure of Pax6/
Pax6(5a) proteins. Phosphorylation sites are marked with an asterisk (*) and the sumoylation site is marked with a number sign (#). The HIPK2 and MAPK phosphorylation sites are
colored blue and yellow, respectively. The amino acid positions correspond to the canonical Pax6 protein. In A and B, the paired domain and the exons that give rise to it are in red,
the glycine-rich linker and the exons that give rise to it are in green, the homeodomain and the exons that give rise to it are in purple, and the PST domain and the exons that give
rise to it are in light blue. (C) Consensus SELEX-driven binding sites of canonical Pax6 paired domain (P6CON), Pax6(5a) paired domain (5aCON) and Pax6 homeodomain (P3), and
site 2-1 which is bound by the homeodomain (HD), the PAI, b-sheets and linker regions. Above the binding sequences are schematic representations of the Pax6 domains that bind
them. N, any nucleotide; W, A or T; S, G or C; K, G or T; M, A or C; Y, T or C.
intricate regulation of the gene, as each promoter may have its own
set of adjacent regulatory sequences, relieving the evolutionary
constraints on each promoter. A third promoter and transcription
start site are located in an intronic sequence between exons 4 and 5.
Termed promoter a (Pa, Fig. 1A), this site initiates transcription of
an alternative transcript, which encodes a truncated Pax6 protein
variant (Pax6DPD, see section 2.2).
Transcription from the above promoters is controlled by various
enhancer sequences with remarkable tissue specificity. Utilizing
sequence conservation, DNaseI-protection assays, in-vitro reporter
transfections and transgenic reporter constructs, several groups
have identified a multitude of regulatory sequences in the Pax6
locus (Kammandel et al., 1999; Kleinjan et al., 2004; Plaza et al.,
1995a; Williams et al., 1998; Xu et al., 1999b), summarized in
Fig. 1A and Table 1.
The cis-elements located upstream of the Pax6 gene and in
introns were initially identified through a study of evolutionarily
conserved regions that were functionally tested using transgenic
Table 1
Tissue-specific Pax6 enhancers.
References
Reporter expression pattern
Approximate pair coordinates
relative to (mouse) P0
Code in Fig. 1
Kammandel et al., 1999
Kammandel et al., 1999;
Williams et al., 1998
Xu et al., 1999b
Kammandel et al., 1999
Xu et al., 1999b
Xu et al., 1999b; Plaza et al., 1995a;
Kammandel et al., 1999
Kleinjan et al., 2004
Pancreatic islets
Pax6 ectodermal enhancer (EE) surface ectoderm
derivatives: cornea, lens, conjunctiva, lacrimal gland
Pancreatic islets, retinal progenitor cells
dorsal telencephalon, hindbrain, spinal cord
Photoreceptor progenitors
Distal retina a, amacrine cells, ciliary body, iris
4.6k
3.9k
a
b
2.3k
1.5e6.5k
3.5k
14k
c
d
e
f
Late eye development
Diencephalon
Rhombencephalon
Pretectum, neural retina and olfactory region
Lens, diencephalon, hindbrain, proximal retina, cerebellum
Telencephalon, neuroretina, retinal pigmented epithelium
Forebrain, diencephalon, pineal gland
17.5k
19k
21k
w105k, 106k and 107k
w110k
w128k
w165k
g
h
i
J,k,l
m
n
o
Griffin et al., 2002
Kleinjan et al., 2006, 2001
Kleinjan et al., 2001
Kleinjan et al., 2006
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
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O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
reporter assays (Kammandel et al., 1999; Williams et al., 1998). Such
analyses demonstrated distinct, yet partly overlapping regulatory
activity, which simulated part of the total pattern of the intact gene.
For example, an enhancer in intron 4 as well as cis-elements
upstream of P0 were found to activate gene expression in the
developing retina (regions f and c in Fig. 1A) (Kammandel et al.,
1999; Xu et al., 1999b), and two distinct regions were identified
as activating reporter expression in the pancreas (regions a and c in
Fig. 1A) (Xu et al., 1999b). Additional regulatory sequences were
found near the proximal P1 promoter, which are important for
expression of the P1 transcript in the CNS and photoreceptors
(Table 1 and regions d and e in Fig. 1A) (Kammandel et al., 1999;
Plaza et al., 1995a; Xu et al., 1999b).
Thus, Pax6 expression in a specific location is mediated by the
combined activity of several transcriptional control elements. This
was exemplified by the decrease, but not elimination, of Pax6
expression upon deletion of a lens enhancer (Dimanlig et al., 2001)
(region b in Fig. 1A). The overlapping activity of regulatory regions
allows intricate regulation of expression levels in defined tissues
and developmental time windows, and also provides redundancy
that contributes to robustness in gene activity. An added benefit of
identifying Pax6 regulatory modules is their use for the establishment of tissue-specific Cre lines such as the transgenes Le-Cre
(including regions a,b,c in Fig. 1A) (Ashery-Padan et al., 2000) and
a-Cre (region f, Fig 1A) (Marquardt et al., 2001), which are widely
used for the study of gene function using Cre/loxP technology in the
lens and retina, respectively.
In addition to the regulatory elements located upstream and
within the introns, more distant cis-elements have been identified
downstream of the gene. These came to light through comprehensive analyses of human aniridia patients who were carriers of
deletions and chromosomal rearrangements (Crolla and van
Heyningen, 2002; Fantes et al., 1995; Lauderdale et al., 2000). The
regulatory elements were located downstream of the most distal
breakpoint reported in human aniridia (SIMO; Lauderdale et al.,
2000; Simola et al., 1983). These elements have been characterized in vivo using human yeast artificial chromosome (YAC) transgenic studies. One such YAC, encompassing 420 kb including the
Pax6-encoding sequence, was able to rescue homozygous Pax6sey/sey
mice, but only when the 30 region of Pax6 was intact (Kleinjan et al.,
2001; Schedl et al., 1996). Interestingly, this 30 region contains
regulatory elements that are functionally conserved between
humans and mice (Tyas et al., 2006). Further analysis of the fulllength and truncated YAC transgenes, evolutionary sequence
comparison and transgenic reporter studies revealed the location
and activity of several novel tissue-specific enhancers (Griffin et al.,
2002; Kleinjan et al., 2006). Additional regulatory elements were
identified in a region that was termed distant downstream regulatory region, located 130 kb downstream of the Pax6 poly-A site
(regions jeo, Fig. 1A) (Griffin et al., 2002; Kleinjan et al., 2001,
2006). Functional study of this downstream regulatory region
proved that it is required for Pax6 expression in some, but not all
tissues (Kleinjan et al., 2006).
Aside from transcriptional regulation, another emerging
mechamism of gene modulation involves microRNAs (Selbach
et al., 2008). Recently, Pax6 was found to be subjected to inhibition by miR-450b-5p in the corneal epithelium (Shalom-Feuerstein
et al., 2012). As the Pax6 30 UTR can be targeted by multiple
microRNAs, this mode of regulation may allow fine tuning of the
levels of expression of Pax6 splice variants.
Taken together, the study of Pax6 regulation provides an excellent paradigm for investigating the regulation of a developmental
control gene (Kleinjan and van Heyningen, 2005). Pax6 encompasses a large control region with numerous complex cisregulatory elements. Some of these sites work in synergy, while
others function redundantly. The challenge now is to understand
how these elements allow for activation of promoters, and to
identify the upstream transcription regulators that enable the
complex spatiotemporal and quantitative regulation of this gene
in vivo.
2.2. Structure and transcriptional activity of the Pax6 proteins
The complex activity of tissue-specific transcription factors,
such as Pax6, is made possible by several functional domains that
facilitate DNA binding and proteineprotein interactions. By alternative promoter usage and splicing, several proteins with different
functional domain combinations can be encoded by the same gene.
In addition, the functions of these proteins may be rapidly modulated by numerous post-translational modifications and
proteineprotein interactions that are triggered by extrinsic cues
and the intrinsic makeup of the cells. Here we summarize the
findings on the activities and modifications of each of the Pax6
protein domains and discuss the implications of these findings in
uncovering the gene networks that are regulated by Pax6.
The most abundant and extensively studied Pax6 variant is the
functional homologue of the Drosophila genes Eyeless (Ey) and Twin
of Eyeless (Toy), referred to as “canonical Pax6” (Czerny et al., 1999;
Quiring et al., 1994). In mammals, this 422-aa protein contains two
DNA-binding domains: a 128-aa bipartite paired domain, which is
shared by all of the PAX proteins, and a 61-aa paired-type homeodomain, which is found in a subclass of PAX genes (Walther and
Gruss, 1991; Wilson et al., 1993). The paired domain and homeodomain are separated by a 78-aa glycine-rich linker. Finally, the Cterminal region of the protein is enriched with proline-serinethreonine (PST) residues that are prone to phosphorylation. The
C-terminal domain plays a role in the transactivation properties of
Pax6 (Fig. 1B) (Czerny and Busslinger, 1995; Glaser et al., 1994;
Mikkola et al., 1999; Tang et al., 1998).
The mammalian Pax6 paired domain is encoded by exons 4e7
(Fig. 1). Crystallographic studies have revealed that the paired
domain of both the human PAX6 and the Drosophila Paired (Prd)
proteins consists of N-terminal and C-terminal subdomains,
termed PAI and RED, respectively, linked by an extended linker
(Fig. 1B). Similar to the structure of a homeodomain, both paired
subdomains fold into three a-helices, two of which form a helixturn-helix (HTH) motif (Xu et al., 1999a, 1995).
Using the systematic evolution of ligands by exponential
enrichment (SELEX) assay with the Pax6 paired domain, Epstein
et al. (1994a) identified its optimal DNA-recognition sequence.
This consensus sequence was termed P6CON (Fig. 1C). Highresolution crystal structure analysis of a complex between P6CON
and Pax6-paired domain revealed that it binds DNA as a monomer.
The DNA binding occurs through interaction with the HTH motifs of
both PAI and RED subdomains, which contact the major groove on
opposing sides of the binding site, while the short b motif N of the
PAI subdomain and the linker subdomain that links PAI and RED
contact the DNA through the minor groove (Xu et al., 1999a). The
PAI subdomain has been found to bind DNA at higher affinity than
the RED subdomain (Epstein et al., 1994a; Xu et al., 1999a), and the
specificity of the Pax6 paired domain has been found to be
dependent on three amino acidsdisoleucine 59, glutamine 61 and
asparagine 64, which are located within the PAI subdomain.
Replacing these residues with the corresponding amino acids of
Pax5 switched the sequence specificity of the domain to that of
Pax5 (Czerny and Busslinger, 1995).
Another form of Pax6 is produced via alternative splicing of exon
5a, which is a 42-bp long segment located between exons 5 and 6
(Fig. 1A). Exon 5a encodes 14 aa, which are inserted within the first
a-helix of the HTH region of the PAI subdomain. This insertion
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
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disrupts DNA binding of the PAI subdomain, while activity of the
RED subdomain is exposed (Fig. 1B) (Epstein et al., 1994b).
Accordingly, the consensus binding sequence identified by SELEX
with the Pax6(5a) paired domain differed from that of Pax6. The
Pax6(5a) consensus sequence is composed of two half sites and
Pax6(5a) binds this sequence as a dimer as each half site is bound
by one protein (termed 5aCON, Fig. 1C) (Epstein et al., 1994b). In
contrast to the Ey and Toy genes, which are analogous to Pax6, the
Drosophila genes Eyegone and Twin of Eyegone show a structural
resemblance to the Pax6(5a) splice variant. Eyegone and Twin of
Eyegone proteins contain only the RED but not the PAI subdomain,
and their DNA-binding specificity is similar to that of the Pax6(5a)
variant (Jun et al., 1998).
In mammals, the ratio between the canonical form of Pax6 and
the Pax6(5a) isoform varies among tissue types. Moreover, patients
who carry a mutation that changes the ratio of the two proteins
demonstrate aniridia and abrogated eye development (Epstein
et al., 1994b; Pinson et al., 2005; Zhang et al., 2001). These findings implicate distinct roles for Pax6(5a) in specific ocular tissues.
In the developing eye, Pax6(5a) is known to play a role in lens and
iris development, based on both gain- and loss-of-function studies
using transgenic mouse models (Davis et al., 2009; Duncan et al.,
2000; Singh et al., 2002). Surprisingly, retinal development seems
unaffected by homozygous Pax6(5a) deletion in mice (Singh et al.,
2002). In contrast to the limited activity of Pax6(5a) in the mouse
retina, this isoform may play an important role in the formation of
the cone-cell-rich fovea in diurnal animals, as Pax6(5a) is highly
expressed in the foveal region, can cause accumulation of foveallike structures upon overexperession in chicks, and is improperly
spliced in humans with isolated foveal hypoplasia (Azuma et al.,
1996, 1999, 2005; Hanson et al., 1999; Vincent et al., 2004).
Exons 8e10 of the Pax6 gene encode a paired-type homeodomain (Fig. 1A), which is found in a large class of homeodomain
proteins (Wilson et al., 1993). Similar to other homeodomains, this
class consists of three a-helices with an HTH motif and an Nterminal arm. However, unlike other types of homeodomains
which bind DNA as monomers, paired-type homeodomains have
been shown to bind cooperatively as homo- or heterodimers to
a palindromic sequence composed of two inverted ATTA separated
by 2 or 3 bp (therefore called P2 and P3, respectively; Fig. 1C)
(Czerny and Busslinger, 1995; Wilson et al., 1993, 1995).
A “Pairedless” Pax6 protein variant (Pax6DPD), which does not
contain the homeodomain, is a result of transcription activity from
the Pa promoter and may also result from alternative splicing
(Carriere et al., 1993; Gorlov and Saunders, 2002; Kim and
Lauderdale, 2006; Kleinjan et al., 2006). Pax6DPD is detected in
some ocular tissues and its overexpression abrogates normal eye
development (Kim and Lauderdale, 2006, 2008). While its physiological activity in the eye is still unknown, overexpression of
Pax6DPD results in microphthalmia, and disrupts lens and corneal
development (Kim and Lauderdale, 2006, 2008). In-vitro and cellculture experiments suggest that Pax6DPD binding to the P3
sequences is dependent on sumoylation of lysine 91 (Yan et al.,
2010). Further studies are required to determine the physiological
roles of Pax6DPD and its sumo-modified forms.
The conserved PST-rich region in the C terminus of Pax6 is
encoded by exons 10e13 (Fig. 1A). This domain has been shown to
function as a transactivator of transcription based on analysis of
mutations and functional studies using transient transfection
assays of GAL4-PST fusion proteins (Czerny and Busslinger, 1995;
Glaser et al., 1994; Mikkola et al., 1999; Tang et al., 1998). Dissection of the PST region showed that its different parts, encoded by
the three different exons, act synergistically and are all needed to
produce the full level of transcriptional activation (Tang et al.,
1998). It was further found that the transactivation activity of the
5
GAL4-PST hybrid is inhibited in a dosage-dependent manner by
free PST domain (Mikkola et al., 1999; Tang et al., 1998). These
results suggested that PST transactivation activity is achieved by
the recruitment of different co-activators to the enhancer via
proteineprotein interactions. However, Pax6 proteineprotein
interactions are not completely dependent on the PST domain:
both the paired domain and the homeodomain have also been
shown to participate in such interactions (Cvekl et al., 1999;
Mikkola et al., 2001).
The C0 terminus of the PAX6 protein may have additional functions to its known role in transcription. A novel Pax6 splice variant,
Pax6(S), was found to regulate the cellular localization of the Ca2þ
channel b 3 subunit (Ca(v)beta; Zhang et al., 2010b). The C0
terminus of Pax6(S) contains a truncated PST domain fused to
a unique amino-acid sequence, which is conserved only in
primates. The expression pattern of this isoform differs from that of
other known PAX6/Pax6 proteins (Zhang et al., 2010b). Investigating the physiological role of Pax6(S) may reveal primate specific
activities of this unique Pax6 variant.
While known mostly for its role as an activator, Pax6 has also
been shown to inhibit the transcription of several genes, such as
members of the bg superfamily of lens fiber cell (LFC)
crystallinsdbB1 crystallin (Crybb1) (Duncan et al., 1998) and gF/gE
crystallins (Cryge,Crygf) (Kralova et al., 2002)das well as the transcription factor Crx in the developing retina (Oron-Karni et al.,
2008). Pax6’s role as a repressor of Crybb1 seems to be independent of the PST domain and appears to be through competition for
promoter occupancy (Duncan et al., 1998).
One approach to addressing the activity of different domains
in vivo is to investigate the phenotype of mouse mutants with point
mutations that alter the activity of specific domains. The allelic
series of the Pax6 gene in mice has been a useful resource for these
types of studies (Favor et al., 2001, 2008; Graw et al., 2005; for an
up-to-date list of alleles go to http://www.informatics.jax.org/
searches/allele_report.cgi?_Marker_key¼12184). A detailed characterization of the cortical phenotype in Pax6 mutants in sequences
encoding paired domain, paired-5a, or homeodomain suggested
distinct roles for each of the domains: for the paired domain in
patterning and specification, for the paired-5a domain in proliferation, and for the homeodomain in boundary formation in the brain
(Haubst et al., 2004).
Although some Pax6 targets were found to be dependent on
a single domain, we need to remember that these domains are not
independent of each other, as mutations in one domain may
abrogate the DNA-binding activity of another. Cooperation between
DNA-binding domains has been shown in the regulation of several
Pax6 targets, including L1, which encodes a neural cell-adhesion
molecule (Chalepakis et al., 1994), the promoter of Gcg, which
encodes Proglocagon (Andersen et al., 1999a; Grapp et al., 2009)
and the promoter of the secreted frizzeled receptor 2 (Sfrp2;
Haubst et al., 2004). Studies of several missense mutants demonstrated that mutation in the PAI subdomain influences functional
properties of the RED subdomain and conversely, that an intact RED
subdomain is required for a functional PAI domain (Chauhan et al.,
2004). It has also been shown that homeodomain activity depends
on a functional paired domain (Mishra et al., 2002). In addition,
both deletions and missense mutations within the Pax6 activation
domain influence Pax6 homeodomain function (Singh et al., 1998,
2000). A recent study demonstrated dynamic regulation of
aAcrystallin (Cryaa). Cryaa in the lens seems to be mediated by the
paired domain (Yang and Cvekl, 2005), whereas in adult olfactory
bulbs, it is dependent on the activity of the homeodomain of
Pax6DPD. Intriguingly, in adult dopaminergic neurons, regulation
of Cryaa is dependent on the activity of Pax6DPD together with that
of the full-length Pax6. These data reveal that the function of Pax6
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
(2012), doi:10.1016/j.preteyeres.2012.04.002
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O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
is dictated by intermolecular interactions between domains within
the protein, as well as by the association of different Pax6 protein
variants (Chauhan et al., 2004; Ninkovic et al., 2010).
The complexity of Pax6 DNA-binding mechanisms and specificity was further uncovered when the two DNA-binding domains
were tested together in vitro. In a SELEX assay, using a peptide
containing both the paired domain and homeodomain of the
Drosophila Prd protein, Jun and Desplan (1996) identified three
different classes of binding sites. The first contained the known
consensus of the Prd paired domain, which resembles P6CON;
a second contained a Prd-type homeodomain-binding site (termed
P2-TAAT NN ATTA); the third, designated PH0, contained both
paired domain- and homeodomain-binding sequences, immediately adjacent to each other. The Prd protein was shown to bind the
PH0 sites as a monomer. These findings suggested three modes of
binding of Prd, and perhaps other homeodomain-containing PAX
proteins, to DNA: (1) through the paired domain, (2) as a dimer
through the homeodomain or (3) as a monomer to the PH0 site
using both DNA-binding domains (Fig. 1C). Combinations between
the two subdomains, PAI and RED, might allow even more binding
variations (Jun and Desplan, 1996). It appears that different
combinations between the three HTH motifs in Pax6 permit
extensive variability in its DNA-binding properties and ultimately,
in the targets that are directly regulated by this gene.
Recently, a SELEX study was conducted using recombinant Pax6
proteins containing either the Pax6 or the Pax6(5a) paired domain
and the homeodomain (Xie and Cvekl, 2011). The enriched
sequences were compared to a list of experimentally validated
Pax6-binding sites, revealing several novel sequences whose
binding to Pax6 was confirmed in vitro using electrophoretic
mobility shift assays (EMSAs). Interestingly, cells transfected with
some of these seqeunces demonstrated high activation of reporter
genes, whereas others actually suppressed expression in the presence of Pax6, indicating that Pax6 repressor activity might occur
through the binding of particular sequences (Xie and Cvekl, 2011).
Three of the novel binding sites contained a homeodomain-binding
sequence next to sequences that are apparently bound by the
paired domain. These sequences were able to activate reporter gene
expression to higher levels than the P6CON sequence alone (site 21, HD/PAI/b/L, Fig. 1C). Two such sites were found in the CR1
enhancer of the mouse c-MAF gene and were shown to be important for its transcriptional activity in the lens (Xie and Cvekl, 2011).
In a separate study, Coutinho et al. (2011) suggested a combined
model of a Pax6-binding site based on bioinformatics analysis of
experimentally validated sites. This model was used for a genomewide search in evolutionarily conserved areas and suggested that
over 200 promoters are regulated by Pax6 in vivo. Several of these
putative targets were confirmed to be dependent on Pax6 expression and to be bound by Pax6 based on chromatin immunoprecipitation (ChIP) analysis.
An interesting attempt to identify novel Pax6-binding sites with
the full-length Pax6 protein was made using cyclic amplification of
protein-binding DNA sequences. These studies revealed novel
binding sites that resemble the consensus rodent B1 repetitive
element (Zhou et al., 2000) and the consensus primate Alu element
(Zhou et al., 2002), both completely divergent from the P6CON site.
These sequences were shown to be bound by Pax6 in vitro using
EMSA (Zhou et al., 2000, 2002). The existence of Pax6-binding sites
on short interspersed sequences (SINEs) such as Alu and B1 offers
a possible evolutionary scenario in which Pax6 “took advantage” of
retro-transposons to recruit new targets (Zhou et al., 2000). It
remains to be seen whether this binding occurs in vivo, thereby
ascertaining its functional significance.
Bioinformatics approaches combined with gene-expression
data and ChIP are imperative for our understanding of Pax6
activity in gene regulation and the ability to predict Pax6 targets
and resolve its gene regulatory networks that mediate the development of multiple eye structures in different species.
Aside from the use of different binding domains, another layer of
complexity in Pax6’s function is achieved through posttranslational protein modifications. Pax6 is a phosphoprotein and
phosphorylation sites have been identified within the PST transactivation domain (Carriere et al., 1993; Mikkola et al., 1999).
Scanning the transactivation domain of zebrafish Pax6, Mikkola
et al. (1999) identified three conserved phosphorylation sites that
are substrates for the mitogen-activated protein kinases (MAPKs)
P38 and Erk2. These phosphorylation sites were not required for
the stability or DNA-binding properties of Pax6, but rather
appeared to play a role in its transactivation properties. One of
these zebrafish Pax6 sites, serine 413, is conserved from sea urchins
to humans and is thus expected to play an important role (serine
398 in mouse, Fig. 1B).
Another kinase shown to enhance the transcriptional activity of
Pax6 via phosphorylation of the transactivation domain is
homeodomain-interacting protein kinase 2 (Hipk2). Three sites that
are primarily phosphorylated by HIPK2 were identified in the
transactivation domain of Pax6: threonine 304, threonine 373 and
threonine 281 (Fig. 1C) (Kim et al., 2006). Co-transfection with
HIPK2 enhanced the transcriptional activity of both the GAL4-PST
hybrid and full-length Pax6 on the Gcg promoter. Furthermore,
p300 was shown to be recruited to the Gcg promoter in a Pax6- and
HIPK2-dependent manner, suggesting that phosphorylation by
HIPK2 enables Pax6 to recruit general transcription factors to its
binding site (Kim et al., 2006). Corresponding with the important
role of phosphorylation in Pax6 activity, the serine-threonine
phosphatase PP1 was shown to dephosphorylate Pax6 in vitro
and in cell culture. The dephosphorylation of threonine 360/serine
361 appeared to inhibit Pax6 activation of the aB-crystallin
promoter in cell culture (Fig. 1C) (Yan et al., 2007).
Two additional post-transcriptional modifications have been
reported for Pax6. The ring finger E3 ubiquitin ligase Trim11 was
shown to affect Pax6 protein stability and transcriptional activity
(Tuoc and Stoykova, 2008). Quail Pax6 and Pax6(5a) were shown to
be targets of O-linked N-acetylglucosamination (O-GlcNAc glycosylation). Putative glycosylation sites were implicated in the paired
domain. This modification was shown not to affect the DNAbinding affinity of the protein, but to potentially affect its
proteineprotein interactions (Lefebvre et al., 2002).
Finally, associations with other transcription regulators also
participate in Pax6’s recognition of DNA targets. Tissue-specific
transcription factors were shown to interact with Pax6 in the
regulation of cell-type genes. In the pancreas, Cdx and Pdx were
shown to interact with Pax6 on Gcg and Somatostatin (Sst)
promoters, respectively (Andersen et al., 1999a,b). During lens
development, Sox2 bound cooperatively with Pax6 to the enhancer
of the chicken d-crystallin gene during initiation of lens development (Kamachi et al., 2001). The binding site, bound cooperatively
by Pax6 and Sox2, differed from P6CON and was not bound by Pax6
alone (Kamachi et al., 2001).
In summary, combinations of Pax6 domains and interactions
with molecular partners may have a major effect on the protein’s
sequence-recognition properties. Accordingly, several of the identified Pax6-binding sites diverge substantially from the consensus
sequences identified for each domain. Moreover, the use of
consensus sequences for in-silico identification of Pax6-binding
sites yields many false positives (Wolf et al., 2009). The challenge
for future research will be to identify the direct targets of Pax6 in
defined developmental contexts and to further resolve the mechanism by which this protein acquires its target specificity in vivo. In
the next section, we look into the known in-vivo functions of Pax6
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
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O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
and the identified targets that mediate this gene’s functions in the
developing eye.
3. The roles of Pax6 in specification and morphogenesis of
the eye
The vertebrate eye is composed almost entirely of the three
ectodermal derivatives of the embryo: the neuroectoderm, the
head SE and the neural crest. Interaction between these embryonic
tissues enables the correct placement and alignment of the individual ocular tissues that together form a functional eye (see Fig. 2
for summary of the morphogenetic and molecular events during
early stages of mouse eye development).
Immediately after gastrulation, the neuroectoderm undergoes
a series of patterning events. As a result, a single patch of ectoderm
in the head region is specified to become the neural part of the eye,
termed "eye field". During neurulation, two extensions from the
eye field evaginate laterally and become the OVs. The OVs then
extend and contact the SE. The area of contact between the OV and
SE develops into a placode of columnar cells, termed the lens placode (LP), and into primordia of other anterior structures such as
the corneal and conjuctival epithelium. The LP and OVs invaginate
simultaneously to give rise to the lens pit and the optic cup (OC,
respectively. The lens pit subsequently detaches from the adjacent
progenitors of the overlying ectoderm to become the lens vesicle.
7
Neural crest cells that migrate between the lens vesicle and the
corneal epithelium will give rise to the corneal stroma. The inner
layer of the OC will give rise to the retinal progenitor cells (RPCs)
while the outer layer contains the progenitors of the ocular pigmented epithelium (PE). The peripheral regions of the OC contain
the progenitors that together with neural crest cells emanating
from the ocular mesenchyme form the auxiliary structures of the
eye: the CB and iris.
The interaction between the OV, SE and surrounding mesenchyme has fascinated developmental biologists, as it is a prime
example of tissue induction, specification and patterning leading to
morphological changes. Below, we explore recent findings on the
transient events and Pax6’s roles during the specification of the
ocular neuroepithelium and lens lineages.
3.1. Specification of OV progenitors
3.1.1. Pax6 is one of several eye-field genes
Specification of the neuroectoderm to the prospective eye is
initiated with the coordinated expression of eye-field transcription
factors (EFTFs) at the anterior neural plate, best characterized in
Xenopus laevis embryos (Li et al., 1997; Zuber et al., 2003). The EFTF
genes include Six3, Lhx2, Rax (Rx), Tbx3, Optx2 (Six6), Nr2e1 (Tlx) and
Pax6, sometimes in conjunction with the head-determination gene
Otx2. These factors are expressed in the eye field with overlapping
Fig. 2. Morphogenesis, molecular pathways and Pax6 regulatory networks during early stages of eye development. (A) Images illustrating the early stages of eye development in
mouse (E8, E9, E10 images adapted from Edinburgh eMouse Atlas Project, Medical Research Council, UK; under the Creative Commons License). Colored areas demonstrate the
Pax6-positive domains of the surface ectoderm derivatives (purple) and neural plate derivatives (green). The neural crest-derived mesenchyme is in blue. (BeE) Summary of the
molecular pathways occurring within the respective stage and tissue and their connection to the Pax6 regulatory network (B, C for the lineages of the neuroectoderm, D, E for the
surface ectoderm lineage). The Pax6 gene networks in C, D and E are provided as supplementary files (supplementary files 1,2,3 respectively) for visualization and analysis by Spike
(http://www.cs.tau.ac.il/wspike/, Elkon et al., 2008; Paz et al., 2011) or cytoscape (http://cytoscape.org/). The genotypes are of the mouse mutants employed to examine Pax6’s role
at the specific stage shown. Abbreviations: FB, forebrain; HM, head mesenchyme; LP, lens placode; OM, ocular mesenchyme; OV, optic vesicle; SE, surface ectoderm; OC, optic cup;
OS, optic stalk; PE, pigmented epithelium; PPR, pre-placodal region; RPCs, retinal progenitor cells. Bar ¼ 100 mm.
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
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O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
temporal and spatial expression patterns and were defined as eyefield genes based on their capacity to induce ectopic eyes in frog
embryos when misexpressed (Zuber et al., 2003). EFTFs co-regulate
each other, as observed by overexpression of different EFTFs in
Xenopus embryos. For example, Pax6 overexpression results in
upregulation of Six3, Lhx2, Tlx and Optx2, while Six3 expression
induces that of Pax6, Tll and Lhx2 (Zuber et al., 2003). Remarkably,
several of the EFTFs (Six3/Six6, Pax6) are homologues of Drosophila
retinal determination genes that comprise a regulatory network
which is essential and sufficient for eye formation in the fly (i.e. sine
oculis/optix, eyeless(ey)/twin of eyeless(toy)), while other EFTFs (i.e.
Rx, Tbx3, Nr2e1) are homologues of Drosophila genes that are also
involved in brain and eye development (i.e. drx, omb, and tll,
respectively; reviewed in Zuber, 2010). Thus the molecular network
involved in early specification of the eye is evolutionarily conserved
in invertebrates and vertebrates, despite the considerable evolutionary distance and marked differences in eye anatomy and
development between the groups.
Pax6 is one of the first EFTFs to be expressed in a wide area of the
neural plate that includes the eye field. Its expression pattern
incorporates those of Rx, Lhx2 and Tbx3 (Zuber et al., 2003). Ectopic
introduction of Pax6 into Xenopus embryos was used to examine
whether Pax6 is sufficient, as in Drosophila, to induce eyes in
vertebrates. While in some experiments Pax6 was only sufficient to
induce lens-like structures without retinas (Altmann et al., 1997),
under other conditions, a fully differentiated ectopic eye could be
induced (Chow et al., 1999). The efficiency of Pax6 as the soletransfected EFTF is much lower than when injected together with
other EFTFs and Otx2 (Zuber et al., 2003). Therefore, in vertebrates,
Pax6’s ability to induce eye structures is limited and dependent on
co-expression of other EFTFs. While the genetic pathway of eyefield determination is probably conserved among vertebrates,
Pax6 activity in other stages of development may not be so well
conserved, even between mammals. A recent study revealed that in
contrast to mouse Pax6, humans PAX6 protein is widely expressed
in the early neural plate during neurulation (Zhang et al., 2010a).
Furthermore, PAX6 was found to be necessary and sufficient for
differentiation of human embryonic stem cells (ESC) to neuroectoderm, while it is dispensable for mouse ESCs (Zhang et al.,
2010a). These findings suggest Pax6 to be generic neuroectodermal specification factor in primates. Thus, in order to
understand the impact of Pax6 on developmental processes, one
must consider there are also species-specific activities that should
be explored.
3.1.2. Roles for EFTFs in the partitioning of the single eye field and
early patterning of the OV
Functional studies of fish and mammalian homologues of
Xenopus EFTFs have revealed their specific and sequential activities
in morphogenesis and patterning of the OV and OC (Fig. 2B).
During neurulation, Sonic hedgehog (Shh) secreted from the prechordal plate induces hypothalamic fates in the central diencephalon and functions in the partitioning of the single eye field
into two OVs (Chiang et al., 1996; Li et al., 1997; reviewed in Byerly
and Blackshaw, 2009). Failure of this midline signaling results in
holoprosencephalyda loss of ventral forebrain structures, and
cyclopiada single optic rudiment, mostly composed of pigmented
cells at the center of the head (Chiang et al., 1996). Six3 plays an
additional role in the patterning of the forebrain as it functions in
a positive regulatory loop that is required for Shh expression in the
diencephalon. Mutations in Six3-binding sites in a Shh enhancer
and a haploid dose of Six3 resulted in Shh loss, holoprosencephaly
and cyclopia (Geng et al., 2008; Jeong et al., 2008). Six3 is required
for the formation of the anterior forebrain and eye, primarily due
to inhibition of Wnt signaling, which is known to antagonize Shh
signaling (Carl et al., 2002; Lagutin et al., 2003; Lavado et al.,
2008).
Following the partitioning of the eye field, the OVs evaginate,
extend laterally and become tightly associated with the overlying
SE. Early studies of OV morphogenesis in the mouse embryo
revealed changes in cell shape during the evagination process
(Svoboda and O’Shea, 1987). More recent studies using live imaging
and single-cell tracking of transparent fish embryos exposed the
importance of different migratory properties of individual RPCs in
relation to the adjacent forebrain (England et al., 2006; Rembold
et al., 2006; reviewed in Martinez-Morales and Wittbrodt, 2009).
At this point, Rx seems to be the essential EFTF for OV morphogenesis in both fish and mammals (Loosli et al., 2003; Mathers et al.,
1997; Medina-Martinez et al., 2009; Rembold et al., 2006; Stigloher
et al., 2006). In fact, Rx seems to control several sequential events
that are eventually required for OV morphogenesis: specification of
the eye field via regulation of Wnt signaling, expression of several
EFTFs (Pax6, Six3, Six6), and regulation of the expression of the Igdomain protein Nlcam, which in fish embryos plays a role in the
migratory properties of RPCs (Brown et al., 2010; Stigloher et al.,
2006; Zhang et al., 2000).
As the OVs invaginate to form OCs, they are already populated
by three distinct progenitor domains, each destined to form
a unique ocular lineage. This early patterning is evidenced by the
restricted expression domains of factors involved in subsequent
differentiation processes. The proximal OV (closer to the neural
tube) expresses the transcription factors Pax2, Vax1 and Vax2,
which are pivotal for formation of the optic stalk (Barbieri et al.,
1999; Hallonet et al., 1999; Mui et al., 2005). Further from the
optic stalk, toward the periphery, the OV contains progenitors of
the PE. These progenitors maintain the expression of microphthalmia transcription factor (Mitf), an essential determinant for
the specification and differentiation of all pigmented cells
(Bumsted and Barnstable, 2000; Nakayama et al., 1998; Opdecamp
et al., 1997; Tassabehji et al., 1994). Finally, the cells populating the
most peripheral OV, which are in close contact with the SE, express
the homeodomain transcription factor Vsx2 (Chx10), which is
subsequently required for proliferation of retinal progenitors and
differentiation of specific retinal neurons (Burmeister et al., 1996;
Liu et al., 1994). Once the OC has formed, additional patterning
occurs. Within the inner OC, nasal/temporal subdivisions are
evident based on expression domains of winged helix transcription
factors Foxg1 and Foxd1 (BF1, BF2; Hatini et al., 1994), while the Tbox and homeodomain transcription factor Tbx5 is detected in the
dorsal OC (Koshiba-Takeuchi et al., 2000). These three genes, as
well as Vax2 expressed in the ventral OC, are involved in establishing the pattern of the retinotectal projections of retinal ganglion
cells (Huh et al., 1999; Koshiba-Takeuchi et al., 2000; Mui et al.,
2002; Schulte et al., 1999; Yuasa et al., 1996).
The patterning of the OV to neuroretinal and pigmented
domains seems to be dependent on several EFTFs. The role of Six3 at
this stage was investigated by deletion of the gene during OV
evagination using Rx-Cre (Liu et al., 2010). Loss of Six3 from the OV
prevented establishment of neuroretinal progenitors. While retinal
fate was not established, the Six3/ optic rudiment was composed
only of pigmented cells. This phenotype has been attributed to
elevated expression of Wnt8b, which was shown to induce PE fate
following misexpression (Liu et al., 2010).
Lhx2 is also required for the establishment of progenitor
domains in the OV. In both somatic and germline Lhx2 mutants,
morphogenesis of the OV takes place normally, but both the PE and
neuroretinal domains fail to form (Hagglund et al., 2011; Porter
et al., 1997; Yun et al., 2009). Interestingly, the role of Lhx2 in the
OV is not mediated by regulation of Rx, Six3 or Pax6 as the
expression of these genes is maintained in Lhx2/ OVs (Hagglund
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et al., 2011). Nevertheless, Lhx2 may function together with other
EFTFs in the regulation of gene expression as Lhx2 is known to form
functional complexes with Pax6 that are required for regulation of
Six6/Otpx2 in the OV (Tetreault et al., 2009).
In Pax6-null mouse embryos, evagination of the OV takes place,
expression of several EFTFs is maintained, and both retinal and PE
progenitors are initially established (Grindley et al., 1995;
Marquardt et al., 2001). The development of the OV is nevertheless abrogated as proliferation in the OV is markedly reduced.
Furthermore, neurogenesis is initiated prematurely based on
upregulation of the pro-neural gene Ascl1 (Mash1), the photoreceptor transcription factor Crx and generic neuronal markers, but
without Pax6, neurogenesis fails to reach completion (Philips et al.,
2005; Oron-Karni et al., 2008, Fig. 2C). Interestingly, establishment
of the neuroretina and pigmented progenitor domains does take
place despite the lack of Pax6 (Baumer et al., 2003; Grindley et al.,
1995). However, analysis of mouse double mutants in both Pax6
and Pax2 genes revealed that both transcription factors are required
for specification of the PE cell fate. Both genes act synergistically to
regulate Mitf and are thus pivotal for specification of the retinal
pigmented epithelium (RPE) lineage (Baumer et al., 2003).
An additional requirement for Pax6 in patterning is establishment of the temporal-nasal and dorsoventral domains of the OV. In
Pax6 germline mutants, the tissue that remains instead of the OV
acquires a ventral fate based on expanded expression of Vax1, while
genes expressed in the dorsal OC (Tbx5) and the temporal/nasal
domains (Foxg1, Foxd1) are not detected (Baumer et al., 2002). A
role for Pax6 in the regulation of Vax1 and Tbx5 was also documented in chick embryos (Leconte et al., 2004). Pax6 is therefore
essential for establishment of progenitor domains and the subsequent differentiation to ocular tissues (Baumer et al., 2002, 2003;
Philips et al., 2005).
To conclude, although Pax6 is expressed at the early neural plate
stage and has a certain capacity to trigger eye induction, loss-offunction studies reveal that unlike Rx, it is not essential for early
OV morphogenesis or specification to a retinal fate, but rather plays
later roles in its patterning, as well as in OC formation and subsequent differentiation to ocular lineages.
3.1.3. Extrinsic cues pattern the OV
The patterning of the OV neuroepithelium is dependent on
growth factors and transcription regulators. Shh secreted from the
ventral midline, which governs eye-field partitioning, also plays
a key role in patterning of the OV. Shh signaling promotes Vax1 and
Pax2 expression ventrally and inhibits the expression of Pax6 and
Rx. Vax1/2 and Pax2 were shown to directly inhibit Pax6 expression
in the ventral OV and Pax6 in turn restricts Pax2 expression to the
prospective optic stalk (Hallonet et al., 1999; Kim and Lemke, 2006;
Mui et al., 2005; Schwarz et al., 1999). Therefore, Shh from the
midline activates optic stalk genes, which in turn inhibit other
ocular lineages.
Studies in chick embryos suggest that the establishment of the
PE and neuroretinal progenitor fates is dependent on the ocular
mesenchyme surrounding the OV and on the lens ectoderm (LE)
overlying the peripheral OV (Hyer et al., 1998; Nguyen and
Arnheiter, 2000). Initially, as the OV extends laterally, low levels
of Mitf are detected throughout the OV. Mitf expression is then
elevated and maintained in the dorsal OV in response to factors that
are secreted from the ocular mesenchyme, transforming growth
factor b (TGFb) family members (bone morphogentic proteins;
BMPs, activin) and Wnt ligands (Fuhrmann et al., 2000; Fujimura
et al., 2009; Grocott et al., 2011; Kagiyama et al., 2005; Muller
et al., 2007; Nguyen and Arnheiter, 2000; Westenskow et al.,
2009). As the OV reaches the SE, the expression of neuroretinal
gene Vsx2 is elevated in these cells. Subsequently, the mutual
9
repression between Vsx2 and Mitf contributes to the establishment
and maintenance of the PE and neuroretinal progenitor domains
(Horsford et al., 2005; Nguyen and Arnheiter, 2000; Rowan et al.,
2004).
The SE itself is thought to provide inductive factors that
promote neuroretinal differentiation. In chicks, removal of the
fibroblast growth factor (FGF)-expressing SE results in reduction
of retinal markers and expansion of the PE domain (Hyer et al.,
1998; Pittack et al., 1997). This phenotype can be rescued by
external FGF administration (Hyer et al., 1998). Accordingly, FGF
appears to suppress Mitf expression and promote Vsx2 expression
(Horsford et al., 2005; Nguyen and Arnheiter, 2000). Important
evidence for FGF signaling in neuroretinal fate was obtained by
conditional inactivation, in the OV, of Shp2, a tyrosine phosphatase that mediates the MAPK-signaling pathway (Van Vactor et al.,
1998). Loss of Shp2 resulted in acquisition of RPE instead of
a neuroretinal fate (Cai et al., 2010). Expansion of the RPE domain
at the expense of the neuroretinal fate was also observed upon
Fgf9 loss (Zhao et al., 2001). These findings support a role for FGF
signaling in OV patterning and implicate SE as the source of the
FGF ligands.
There are, however, additional findings that question the notion
of the lens SE being essential for neurogenesis. Somatic Pax6
deletion from the LE using the Le-Cre transgene (see section 3.2.3)
prevents lens development but not the establishment of neuroretinal and RPE domains or the eventual invagination of the OC and
retinogenesis (Ashery-Padan et al., 2000; Smith et al., 2009). In that
mutant, the morphology of the OC is abrogated, as there are several
folds instead of a single cup. Nevertheless, earlier deletion of Pax6
using Ap2a-Cre did arrest OC morphogenesis, suggesting an early
role for Pax6 in pre-placode SE in the patterning of the adjacent OV
(Smith et al., 2009).
A recent groundbreaking experiment further explored the
contribution of external cues to OC patterning (Eiraku et al.,
2011). The authors induced OC formation from cultured mouse
embryonic stem cells in a three-dimensional growth environment. From an initially undifferentiated sphere, Rx-positive OVs
evaginated outwards. These OVs spontaneously established
a distal-proximal axis, in which neuronal markers were found
distally and RPE-like markers were expressed proximally.
Remarkably, the proximal RPE layer acquired structural rigidity,
while the distal more flexible layer of the OV spontaneously
invaginated and formed a two-layered OC resembling the normal
embryonic OC. The layers of stem-cell-derived OC further
differentiated into a fully pigmented RPE and a neuroncontaining retina (Eiraku et al., 2011). Further characterization
of OC morphogenesis in this culture system is required to
determine the full extent of the self-organizing properties.
Nevertheless, analyses conducted to date suggest that growth,
patterning, morphogenesis and differentiation of the OV are at
least partly intrinsic and do not require external cues from
tissues such as the SE, which was absent in this experiment
(Eiraku et al., 2011). This suggests that the LE does not provide
instructive cues for the adjacent OV but rather plays a restrictive
role in preventing surrounding tissue signals from reaching the
distal OC. The study by Eiraku et al., further demonstrated that
the patterning of the cultured OV and its subsequent morphogenesis require adjacent Rx cells, and that the activity of these
cells could be replaced by Wnt3b (Eiraku et al., 2011). More
studies are required to further characterize the identity of the
Rx cells that may function in promoting RPE fate in the culture
system instead of ocular mesenchyme. This relatively simple
culture system is expected to provide novel insight into the
mechanisms governing tissue interactions during organogenesis
of the eye in mammals.
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3.2. The multiple functions of Pax6 in the development of OC
derivatives
3.2.1. The development of the OC neuroepithelium to the ocular
lineages
The progenitors that populate the OV will differentiate gradually, after OC formation to the neuronal and non-neuronal ocular
tissue types: the neuroretina, the RPE, the CB and the iris. The
neuroretina consists of three cell layers separated by two synaptic
regions (Dowling, 1987). The cone and rod photoreceptors populate
the outer nuclear layer; the bipolar, amacrine and horizontal
interneurons and Muller glia reside in the inner nuclear layer; the
ganglion cell layer is populated by ganglion cells, as well as
a subtype of displaced amacrine interneurons. In close contact with
photoreceptors is the RPE, a single layer of cuboidal epithelium that
is essential for the development, homeostasis and activity of the
photoreceptors (Strauss, 2005). Anterior to the retina and RPE are
the CB and iris, which are components of the anterior segment of
the eye. The CB secretes components of the aqueous humor, the
vitreous and inner limiting membranes, and is vital for the maintenance of ocular pressure and for survival of the ganglion cell layer
(Gould et al., 2004; Halfter et al., 2005, 2008). Anterior to the CB is
the iris, which contains the inner pigmented layer and the overlying muscle tissue. The iris muscle controls pupil size and thus the
amount of light entering the eye (reviewed in Cvekl and Tamm,
2004; Davis-Silberman and Ashery-Padan, 2008). Aside from
these physiological activities of the CB and iris, a population of
pigmented cells of the CB and iris appear to exhibit progenitor cell
properties as they proliferate and give rise to neural spheres in
culture, although with limited potential for differentiation to
retinal neurons (Ahmad et al., 2000; Asami et al., 2007; Cicero et al.,
2009; Gualdoni et al., 2010; Lord-Grignon et al., 2006; Sun et al.,
2006; Tropepe et al., 2000).
The differentiation of these highly specialized ocular tissues
from the neuroretinal and pigmented progenitor domains occurs
gradually, following OC formation. Neurogenesis and RPE differentiation are initiated at the center of the OC, close to the optic
stalk, progressing toward the periphery and arresting at the most
peripheral tips of the OC, where the non-neuronal progenitors of
the CB and iris reside (Fig. 3, Davis-Silberman and Ashery-Padan,
2008; Hu and Easter, 1999). Morphogenesis and differentiation of
the CB and iris are triggered in mice close to birth and are
completed upon opening of the eyelids, around postnatal day 10
(P10, reviewed in Cvekl and Tamm, 2004). It is thus evident that
multiple developmental processes occur simultaneously in the
inner OC, including central to peripheral progression of neurogenesis and RPE differentiation, as well as the establishment of
non-neuronal progenitors at the peripheral OC. The molecular
mechanisms that participate in establishing the CB and iris
progenitor pools and the roles of Pax6 in their differentiation have
been recently reviewed (Davis-Silberman and Ashery-Padan,
2008). Here we focus on the molecular events of retinal neurogenesis, and Pax6’s role in this process.
All retinal neurons and the Muller glia are derived from multipotent RPCs that populate the pseudo-stratified neuroepithelium of
the inner OC (Holt et al., 1988; Turner and Cepko, 1987; Turner et al.,
1990; Wetts et al., 1989; Wong and Rapaport, 2009). RPCs gradually
change their competence to differentiate to different cell types
which are therefore generated in a partly overlapping temporal
order, an order that is conserved among vertebrate species (CarterDawson and LaVail, 1979; Cepko et al., 1996; Harman and Beazley,
1987; Holt et al., 1988; Rapaport et al., 1996, 2004; Stiemke and
Hollyfield, 1995; Young, 1985). Early-born cell types include
retinal ganglion cells, horizontal cells, cone photoreceptors and
amacrine cells. Bipolar and Muller glia cells are born last. Rod
photoreceptors, which make up most of the retinal cells, are born in
parallel to the other cell types (Carter-Dawson and LaVail, 1979;
Morrow et al., 1998). As newborn precursors differentiate, they
migrate either basally (closer to the lens) or apically (adjacent to
the RPE; Fig. 3) (reviewed in Baye and Link, 2008). In parallel to cell
differentiation, new progenitors are continuously being generated
in the proliferative zone known as the neuroblastic layer (Fig. 3).
Neuronal differentiation, synaptic connectivity and lamination are
completed in the mouse eye after birth (reviewed in Mumm et al.,
2005; Reese, 2011).
Several transcription factors are expressed in the OV and early
OC, including Rx, Pax6, Six3, Six6, Lhx2, Chx10 and Sox2. These genes
seem to be required for proliferation and thus expansion of the
progenitor pool (reviewed in Agathocleous and Harris, 2009). The
activity of these factors is not restricted to cell proliferation, as they
are also detected and function in sub-types of differentiated
neurons: Pax6 is required for differentiation of most retinal lineages, Chx10 for the bipolar cell fate and Rx in promoting Muller glia
and photoreceptors (Burmeister et al., 1996; Furukawa et al., 2000;
Marquardt et al., 2001). The activity of Pax6 and Chx10 in cell-fate
acquisition appears to be dependent on co-expression with basic
helix-loop-helix (bHLH) pro-neural genes, which are known to
mediate cell-fate choices throughout the CNS (Hatakeyama et al.,
2001; Inoue et al., 2002; Ohsawa and Kageyama, 2008). A major
challenge for studies on the functions of retinal progenitor genes
will be to distinguish their roles in cell proliferation from their
activity in cell fate specification and differentiation.
Corresponding with the central to peripheral progression of
neurogenesis in the retina, the factors that regulate the timing of
cell differentiation are differentially expressed along the centralperipheral OC (Fig. 3B). One example of a signaling pathway that
functions within the differentiation zone is the Notch pathway. The
Notch target Hes5 is expressed in the neuroblastic layer in a pattern
that follows the central-to-peripheral wave of differentiation
(Yaron et al., 2006). Notch is required for maintaining the RPC pool
by inhibiting pro-neural gene expression and neuronal differentiation. In the retina, this pathway also functions in facilitating retinal
diversity by selective suppression of ganglion and conephotoreceptor cell type fates (Cau and Blader, 2009; Jadhav et al.,
2006; Riesenberg et al., 2009b; Yaron et al., 2006). Another
example is Shh which, in the OC, seems to function within the
differentiation zone. The Hedgehog ligand gene Shh and its target
Gli1 are expressed in a central-to-peripheral pattern corresponding
with the differentiation wave (Wang et al., 2005). Conditional
deletion from the RPCs of Shh or Smoothened (Smo), a component
of hedgehog signaling, revealed that this pathway is required for
maintaining the pool of RPCs and for the specification of late-born
retinal lineages (Sakagami et al., 2009; Wang et al., 2005; Wallace,
2008). The main effector of Hedgehog signaling in the embryonic
retina is the transcription factor Gli2. Interestingly, Gli2 was found
to be a direct transcriptional activator of Hes1, a known target of the
Notch pathway in retinogenesis (Wall et al., 2009). These findings
reveal that the two signaling pathways converge in regulating the
timing of cell differentiation and in determining cell fate in the
course of retinal development in mammals.
Suppressor of fused (Sufu) inhibits the Hedgehog pathway and
thus opposes the Shh signals emanating from the differentiating
ganglion and amacrine cells. Sufu was recently shown to inhibit the
activity of Gli2 during retinogenesis. Deletion of Sufu from proliferating retinal progenitors initially leads to increased proliferation,
followed by premature cell-cycle exit and loss of expression of the
retinal determination genes Rx, Pax6 and Vax2. The Sufu-deficient
RPCs differentiated into aberrant-type interneurons partially
resembling amacrine cells based on expression of Gad67 and syntaxin (Cwinn et al., 2011). Several additional factors are expressed
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11
Fig. 3. Pax6 roles in the optic cup (OC) neuroepithelium. (A) The differentiating neurons are viewed by antibody labeling of the carbohydrate epitope VC1.1 (green). At E12.5,
differentiating ganglion cells are detected in the central OC and their exons extend and form the optic nerve. The cells in the peripheral OC have not yet begun to differentiate,
corresponding with the central-to-peripheral progression of the differentiation front (labeled with arrows). Pax6 protein distribution was visualized using antibody labeling (red in
A). In the OC progenitors, Pax6 is distributed in a gradient; Pax6 expression is high in the peripheral regions of the OC and low in the central regions of the OC. (B) Scheme of
expression of several genes whose activity correspond with the central-to-peripheral regionalization of the OC: Hes5, Gli1 and Sfrp2 are detected in progenitors within the
differentiation zone, while Wnt2b, SSEA1, Otx1, Sufu and Pax6high are detected in peripheral retinal progenitor cells (RPCs) as well as non-neuronal progenitors destined to ciliary
body (CB) and iris fates. (C) Summary of Pax6 activity in each of the three regions of the inner OC; in central RPCs, where differentiation is initiated, in peripheral RPCs with delayed
initiation of differentiation and in the non-neuronal progenitors destined to CB and iris fates. Abbreviations: PE, pigmented epithelium; NNP, non-neuronal progenitors; CO, cornea;
GCL, ganglion cell layer; NBL, neuroblastic layer; ON, optic nerve; ONL, outer nuclear layer. Scale bar in A ¼ 50 mm.
in the peripheral OC and have been implicated in promoting nonneuronal CB and iris fates, as well as opposing the differentiation
front. The Wnt pathway has been extensively investigated in this
context, as Wnt/b-catenin reporter activity and expression of
pathway components were detected in the OC periphery. Canonical
Wnt signaling seems to promote non-neuronal fates and has been
implicated in regulating the rate of retinal cell differentiation (Cho
and Cepko, 2006; Kubo et al., 2003, 2005; Liu et al., 2003, 2007;
Ouchi et al., 2011). Stage-specific embryonic antigen gene 1
(Ssea1, or Lewis X carbohydrate), which is a downstream target of
Wnt, was detected exclusively in late-differentiating RPCs of the
peripheral OC and in the progenitors of the CB and iris (Koso et al.,
2006). Ssea1 expression was not detected in the differentiated
neurons and was only retained in the CB and iris (Koso et al., 2006).
Another non-neuronal marker in the retina is Otx1, which is initially
detected in the peripheral OC and becomes gradually restricted to
the non-neuronal progenitors (Fig. 3) (Martinez-Morales et al.,
2001). Otx1 is required for CB differentiation and displays functional redundancy with Otx2 in the differentiation of various OC
derivatives (Acampora et al., 1996; Martinez-Morales et al., 2001).
Expression of progenitor genes such as Otx1 and Ssea1 in the
peripheral OC suggests that the partitioning of neuronal and nonneuronal progenitor domains is a gradual process that accompanies the maturation of RPCs and patterning of the inner OC.
3.2.2. Roles for Pax6 in the differentiation of OC progenitors
Pax6 is required for OC formation and plays a pivotal role in the
development of all OC sub-structures. Its pleiotropic activity can be
deduced from its dynamic expression pattern. Pax6 is initially
expressed throughout the OC, including RPCs in the inner OC and
PE progenitors in the outer layer. Once differentiation is initiated,
expression becomes restricted: it is reduced in the differentiated
RPE but persists in the iris and CB epithelium to adulthood (DavisSilberman et al., 2005). In the inner OC, Pax6 expression is higher in
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the RPCs located in the peripheral OC, which are late to differentiate, and is lower in progenitors located centrally within the neural
differentiation zone (Fig. 3, Davis-Silberman et al., 2005). This
complex pattern is partly mediated by the a-enhancer of Pax6 (Fig.
1A, f). This regulatory region, which is negatively regulated in the
ventral OC by the transcription factors Vax 1/2 (Section 3.1.2, Mui et
al., 2005) is essential for the elevated expression of Pax6 in the
nasal and temporal domains of the peripheral OC and is sufficient to
direct reporter gene expression to these regions (Baumer et al.,
2002; Kammandel et al., 1999; Marquardt et al., 2001). Pax6 is
expressed in all mitotic progenitors during retinogenesis: its
expression is lost in post-mitotic precursors of photoreceptors and
bipolar neurons but is maintained in differentiated amacrine,
horizontal and ganglion cells (Hsieh and Yang, 2009; Macdonald
and Wilson, 1997; Oron-Karni et al., 2008).
Several roles are attributed to Pax6 in the course of differentiation of the various OC derivatives. These functions were exposed
by a Cre/loxP mutagenesis employing the a-Cre, Chx10-Cre and DctCre transgenes (Fig. 3, Davis et al., 2009; Davis-Silberman et al.,
2005; Marquardt et al., 2001; Oron-Karni et al., 2008). First, Pax6 is
required to specify CB and iris progenitor pools, as neither CB nor
iris were formed when Pax6 was deleted from the distal OC with
either a-Cre or Dct-Cre (Davis et al., 2009; Marquardt et al., 2001).
Second, Pax6’s role in establishing the iris progenitor pool is dosedependent, as somatic inactivation of a single allele of Pax6 in the
peripheral OC leads to a smaller progenitor pool and eventually, iris
hypoplasia (Davis et al., 2009; Davis-Silberman et al., 2005). Lastly,
loss-of-function and rescue experiments infer a role for the
canonical Pax6 isoform in iris growth and differentiation (Davis
et al., 2009; Singh et al., 2002).
Aside from its roles in iris and CB differentiation, a dual role for
Pax6 has been identified in the RPCs. The different activities of Pax6
in these cells seem to correlate with the location of the RPCs along
the central-to-peripheral axis of the OC and thus with their
differentiation potential. The RPCs at the peripheral OC, which are
late to differentiate, require Pax6 for completion of neurogenesis,
for repression of Crx and proliferation (Figs. 3 and 4). Thus, upon
Pax6 loss, these cells do not proliferate: they upregulate Crx but fail
to differentiate to mature photoreceptors (Oron-Karni et al., 2008).
Crx, as well as its regulator Otx2, are the earliest markers for
photoreceptor precursors and both are essential for photoreceptor
differentiation and function (Chen et al., 1997; Freund et al., 1997;
Furukawa et al., 1997; Swain et al., 1997; reviewed in Hennig
et al., 2008). Notably, the elevation of Crx in the Pax6-deficient
RPCs seems to be independent of Otx2 as the latter is not expressed
in Pax6-deficient RPCs. Moreover, in the Pax6/ optic rudiment,
premature expression of Crx is evident during early stages of retinal
development (embryonic day (E) 9.5, not shown). Thus, during the
early proliferative stages, the RPCs require Pax6 to inhibit cryptic
expression of Crx and premature onset of abrogated neurogenic
program.
In contrast to the role of Pax6 in the RPCs populating the
peripheral OC, the RPCs located more centrally, closer to the
differentiation front, no longer require Pax6 for their neurogenic
potential but rather for their multipotency. Thus, when Pax6 is
inactivated in these cells, they do not form most of the retinal
lineage. Instead, they differentiate exclusively into sub-classes of
amacrine interneurons (Fig. 4) (Marquardt et al., 2001; Oron-Karni
et al., 2008). Corresponding with the findings in mice, reducing
Pax6 activity in frog embryos using a dominant-negative Pax6
transgene elevates the number of GABAergic interneurons
(Zaghloul and Moody, 2007a,b).
The differential response of RPCs to Pax6 loss reveals innate
differences in the intrinsic potential of the distal and proximal
Fig. 4. Phenotypes of the retinal neuroepithelium following Pax6 loss. (A) At E14 the expression of Crx is detected by in situ hybridization in the photoreceptor precursors
populating the apical optic cup (OC). (B) In Pax6 systemic knockout embryos (Pax6lacZ/lacZ), cryptic expression of Crx is detected in the optic rudiment, although these cells do not
differentiate into mature neurons. (C) Somatic deletion of Pax6 (Pax6loxP/loxP;a-Cre) from the OC results in elevation of Crx in the peripheral OC. These CrxþPax6 cells do not express
additional neuronal markers and are not detected in the retina after birth. The Pax6 RPCs located more centrally do not express Crx and eventually differentiate to GABAergic
amacrine interneurons which are Gad67þ (red, E). (D) In the control retina, Gad67 is detected in about 40% of the amacrine interneuron subtypes. Cells located distally to the dashed
line in C are deficient for Pax6 due to a-Cre-mediated deletion (not shown). Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblastic layer; ONL, outer
nuclear layer. Scale bar in A ¼ 100 mm and refers to AeE.
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RPCs, which is in agreement with the different molecular composition observed between RPCs along the central-peripheral axis of
the OC and with the gradual onset of differentiation (Fig. 3). The
different response of the OC progenitors to Pax6 loss seems to be
irrespective of onset of Cre activity as differential response of RPCs
to Pax6 loss was also evident when using the Chx10-Cre, which is
active in both central and peripheral regions of the OC from around
E10.5 (Oron-Karni et al., 2008; Rowan and Cepko, 2004). Interestingly heterogeneity among multipotent RPCs was detected in
embryos completely devoid of Pax6 (Pax6lacZ/lacZ; St-Onge et al.,
1997). In these Pax6/ mutants, the optic rudiment contained
Crxþ; VC1.1 and Crx; VC1.1þ cells (Oron-Karni et al., 2008). This
suggests that early on, prior to onset of cell differentiation, RPCs are
heterogeneous in their differentiation potential and in the role Pax6
plays in them.
The loss of multipotency following Pax6 loss in central RPCs
was attributed to reduced expression of several bHLH transcription factors, which are known to promote neuronal specification
(Hatakeyama and Kageyama, 2004; Marquardt et al., 2001).
Accordingly, several pro-neural transcription factors were shown
to be dependent on Pax6 in the developing brain and endocrine
cells of the pancreas. Enhancer elements of the bHLH factors Ngn2,
Mash1, Math5 and Neurod1 were found to be bound by Pax6,
suggesting that it directly regulates their expression (Gosmain
et al., 2010; Marquardt et al., 2001; Riesenberg et al., 2009a;
Visel et al., 2007). In contrast to pro-neural bHLH factors, antineural bHLH under the control of Notch signaling helps maintain
the progenitor pool of the neuroepithelium (reviewed in
Hatakeyama and Kageyama, 2004). Removal of Notch receptor in
mouse embryonic retina results in loss of the progenitor pool and
early differentiation into a specific fate of cone photoreceptors
(Jadhav et al., 2006; Yaron et al., 2006). While there is substantial
evidence that Pax6 is required for the expression of pro-neural
transcription factors, it is currently unclear how it influences the
expression or activity of Notch-pathway components. The intricate
regulatory interactions between Pax6 and Notch needs to be
resolved in future studies.
In addition to its activity in cell-fate acquisition through
regulation of transcription factors, there are multiple findings
supporting a role for Pax6 in the regulation of cell adhesion.
Studies of Pax6 mutants have suggested alterations in the
expression of cell-adhesion molecules and in the extracellular
matrix (ECM) mediating contact between the OV and SE (Collinson
et al., 2000; Huang et al., 2011). In the spinal cord, Pax6 is required
for the expression of N-CAM. In the developing cerebral cortex,
Pax6 was found to regulate expression of the cell-adhesion
molecule R-cadherin and ECM molecules, which are required for
boundary formation and axon outgrowth (Andrews and Mastick,
2003; Gotz et al., 1998; Holst et al., 1997; Stoykova et al., 1997).
As a consequence of Pax6 knockdown in Xenopus embryos, the
homologue of R-cadherin, N-cadherin, as well as other celladhesion molecules, were shown to be downregulated (RunggerBrandle et al., 2010). Studies on Pax6 mutants have further
revealed that loss of Pax6 disturbs cytoskeletal organization, thus
affecting cell migration and neurite outgrowth in the cerebellum
(Engelkamp et al., 1999; Yamasaki et al., 2001). In the mouse
retina, d-catenin was found to be a downstream target of Pax6
(Duparc et al., 2006). d-catenin is known to play a role in neurite
morphology and thus could mediate Pax6 activity during neuronal
differentiation (Martinez et al., 2003). In support of this, abnormal
growth and navigation errors of retinal axons have been documented in mice overexpressing Pax6 (Pax77; Manuel et al., 2008).
Future functional studies of Pax6’s role in post-mitotic neurons are
required to further substantiate this gene’s activity in neurite
extension and axon growth and guidance.
13
3.2.3. Pax6 in RPC cell-cycle regulation
In the developing nervous system, cell-cycle length and the
balance between cell-cycle exit and re-entry are crucial for the
eventual production of the correct number and types of differentiated cells (Agathocleous and Harris, 2009; Farkas and Huttner,
2008). In addition to its role in cell-fate acquisition, Pax6 has
been implicated in the regulation of cell-cycle kinetics in embryonic progenitors and adult neural stem cells (Georgala et al., 2011;
Osumi et al., 2008). In the developing telencephalon, Pax6 is
required for normal interkinetic nuclear migration, as mispositioning of nuclei of cells in the S-phase and M-phase was
documented in the Pax6 mutant brain (Estivill-Torrus et al., 2002;
Gotz et al., 1998; Warren et al., 1999). Interestingly, distinct roles
were attributed to each of Pax6’s splice isoforms in cortical proliferation and specification: its canonical form participates in both
proliferation and specification while Pax6(5)a is primarily involved
in cell proliferation (see section 2, Haubst et al., 2004).
The alteration in cell-cycle parameters following Pax6 loss varies, depending on the developmental context. In early cortical
progenitors, a marked increase in the number of cells in the S-phase
was detected in Pax6 germline mutants (Estivill-Torrus et al., 2002;
Gotz et al., 1998; Warren et al., 1999), accompanied by premature
neuronal differentiation (Quinn et al., 2007). In contrast, in the
diencephalon of mice and in the spinal cord of chick embryos,
a marked reduction in the number of cells in the S-phase was
observed following loss of Pax6 (Bel-Vialar et al., 2007; Warren and
Price, 1997). Significant reduction in cell proliferation was also
found in the optic rudiment of mouse Pax6 germline mutants. This
was further accompanied by premature expression of differentiation markers, though precursors failed to differentiate into known
retinal cell types (Philips et al., 2005; Oron-Karni et al., 2008). In
accordance with the finding in mice, reduced cell proliferation was
also detected in frog retina following Pax6 knockdown (Zaghloul
and Moody, 2007a,b). Furthermore, Pax6 was found to be required
for the proliferation and expansion of neurospheres obtained from
the mammalian CB (Xu et al., 2007). Seemingly conflicting findings
were obtained in Pax6 cells isolated from OVs and grown in culture
(Duparc et al., 2007): under the culture conditions used, Pax6
mutant cells seemed to proliferate more extensively than cells
isolated from control OV (Duparc et al., 2007). It may be that
dissociation and culturing change the cells’ response to Pax6 loss.
Several mechanisms have been suggested for the involvement of
Pax6 in cell-cycle regulation. First, the downstream targets of Pax6
that play a role in differentiation may be involved in the regulation
of cell-cycle kinetics (Farah et al., 2000; Feng et al., 2010;
Ochocinska and Hitchcock, 2009). Second, recent studies
comparing the transcriptome of Pax6-positive and negative cortical
progenitors have shown altered expression of various cell-cyclerelated components such as Ccnd1, Ccnd2, and Cdkn1c (P57Kip2)
(Estivill-Torrus et al., 2002; Holm et al., 2007). Moreover, Pax6
protein may also be involved directly in cell-cycle regulation
through binding to the cell-cycle inhibitor Rb. This possibility is
based on the finding of Pax6 binding to hypo- but not hyperphosphorylated Rb (Cvekl et al., 1999).
In addition to the regulation of cell-cycle genes, other mechanisms have been proposed for Pax6 involvement in cell-cycle
kinetics. Direct imaging of nuclear movement and centrosome
behavior by time-lapse video microscopy in control and Pax6/
embryonic rat cortex infers a role for Pax6 in the positioning of the
centrosomes (Tamai et al., 2007). In another study conducted in cell
culture, Pax6 was suggested to play a role in sister-chromatid
separation (Zaccarini et al., 2007). Finally, in a recent study, the
microtubule-associated protein Spag5 which regulates orientation
of cleavage in progenitor cells was found to be a direct transcriptional target of Pax6 in neural progenitor (Asami et al., 2011).
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It thus appears that Pax6 has multiple converging activities in
the transcriptional regulation of cell-cycle regulators, as well as
direct effects on the machinery of cell division. The challenge in
future studies will be to elucidate how these activities integrate
with Pax6’s role as a determinant of cell fate.
3.3. Specification of the ocular surface ectoderm
3.3.1. Pax6 determines the lens pre-placodal region
Following early studies by Spemann (1901) on lens induction, it
was long thought that contact with the SE is the primary inductive
signal for the SE to form a placode, facilitating eye morphogenesis.
However, later studies conducted in amphibians and avian embryos
revealed that lens induction is a multi-step process, initiated prior
to formation of the OV (Grainger, 1992; Saha et al., 1989; Sullivan
et al., 2004). This process begins as head SE cells are first specified to the pre-placodal region (PPR). The PPR contains the
precursors to all sensory placodes, including the lens, the olfactory
and otic placodes, and sensory cranial ganglia (Streit, 2007).
It is interesting to note that the different segments of the PPR
express different members of the PAX gene family in an anteriorposterior order. Pax6 is expressed in the areas that will become
the adenohypophysis, olfactory and LPs, Pax3 in the trigeminal
placode and Pax2 and Pax8 in the otic (inner ear) and trigeminal
placodes. Despite this patterning, all pre-placodes are first biased to
become a lens. Primary explants of chick pre-placode segments
isolated from embryos prior to neurulation at HH stage 6
(Hamburger and Hamilton, 1951) spontaneously expressed lens
markers, including Pax6 and crystallins, and formed lens-like
structures (Bailey et al., 2006). This was true for ectodermal
segments from the head, but not from the trunk region (Sullivan
et al., 2004). Therefore, there is strong evidence that a large area
of the anterior head ectoderm, which largely corresponds to the
PPR, is specified to a lens fate long before induction by the evaginating OV.
The expression pattern and regulation of Pax6 in the PPR and the
eye field support the notion that these regions are specified independently. Prior to closure of the anterior neural tube, the SE is
located lateral to the neural primordium. In the mouse at this stage
(E8), the Pax6 expression domain seems to encompass both
neuronal and non-neuronal ectodermal progenitors (Fig. 2A, E8)
(Grindley et al., 1995). However, in the chick embryo, where the
early stages of development are easier to monitor, Pax6 is first
expressed in a continuous, crescent-shaped area of ectoderm
around the presumptive neural plate at HH stage 8 (4 somites)
(Bhattacharyya et al., 2004; Li et al., 1994). This domain is separate
from expression in the anterior neural plate, which becomes
evident at around HH stage 10. This crescent largely corresponds to
the anterior PPR, a region that will form the olfactory and ocular
placodes (Bhattacharyya et al., 2004). Therefore, at least in the
chick, it seems that Pax6 is induced independently in the PPR, and
earlier than its expression in the neural plate.
Consistent with independent activation of Pax6 in the PPR and
eye field, several studies of mouse mutants have revealed that
different gene networks control Pax6’s expression in the lens SE
and neuroectoderm. One major difference is that in the LE, but not
in the OV, Pax6 positively auto-regulates its own expression. This
became evident in Pax6sey/sey embryos, where Pax6 protein was
not detected anywhere in the SE, although expression of the
mRNA transcript was maintained in the OV itself (Grindley et al.,
1995). Further support for cell-autonomous auto-regulation of
Pax6 in the head SE is the reduced expression of GFP reporter from
the Le-Cre transgene following Pax6 somatic loss from the SE. The
Le-Cre transgene contains Pax6 lens regulatory regions that are
directly regulated by Pax6 itself (Aota et al., 2003; Ashery-Padan
et al., 2000). Besides being auto-regulated, Pax6 expression in
the PPR is regulated by Six3. This regulation is highly dynamic
throughout lens development. Six3 is expressed prior to Pax6 in
both the PPR and the eye field, but soon afterwards a positive
regulatory loop exists between Six3 and Pax6. In contrast, Six3 and
Pax6 do not regulate each other in the OV (Ashery-Padan et al.,
2000; Goudreau et al., 2002; Liu et al., 2006b, 2010; Marquardt
et al., 2001). This further demonstrates that the gene network
that regulates Pax6 expression in the LE is different from that in
the neuronal eye field.
The timing and level of expression of Pax6 must be tightly
regulated, as altering these parameters abrogates eye development
(Davis-Silberman et al., 2005; Davis et al., 2009; Duncan et al.,
2004; Schedl et al., 1996; van Raamsdonk and Tilghman, 2000;
see section 3.4). The Pax6 ectodermal enhancer (EE, Table 1b, Fig.
1A) has been shown to mediate Pax6 expression levels in the
PPR. This region of only 107 bp is bound by several transcription
factors, including Pax6 itself, Six3, Sox2, Oct1(PouF1) and homeoproteins of the TALE superfamily Meis1, Meis2 and Prep1 (pKnox1)
(Aota et al., 2003; Donner et al., 2007; Liu et al., 2006a; Rowan et al.,
2010; Zhang et al., 2002). Detailed biochemical and transgenic
studies revealed that transcriptional synergy of the Prep1-bound
sites regulates the timing and levels of expression of Pax6 within
the LE (Rowan et al., 2010). Taken together, these studies on the EE
of Pax6 provide important insight on how the affinity and synergistic activity of binding sites, as well as interactions between
different transcription factors, control the timing and levels of gene
expression during organogenesis.
3.3.2. Extrinsic cues in lens-fate determination
The lens-forming competence of the PPR, which initially
encompasses most of the head ectoderm, becomes rapidly
restricted to only the lens pre-placode ectoderm that overlies the
OV (Fig. 2A). Prior to morphogenesis of the LP itself, the lens PPR
expresses a unique combination of factors, including Pax6, Six3,
Sox2, FoxE3, BMP7 (a ligand of the TGFb super-family) and Sfrp2 (an
inhibitor of the Wnt pathway). These genes demarcate the lens
primordium prior to LP formation (Furuta and Hogan, 1998;
Kamachi et al., 2000; Wawersik et al., 1999). Among these genes,
Pax6, Six3 and Bmp7 are essential for formation of the LP (AsheryPadan et al., 2000; Furuta and Hogan, 1998; Liu et al., 2006b;
Smith et al., 2009; Wawersik et al., 1999). Studies conducted in
the last two decades have revealed several restrictive and inductive
signals from the surrounding tissues that define the eventual
location of the LP.
3.3.2.1. The neural crest inhibits lens fate through negative regulation
of Pax6. Due to the early specification of the PPR and the wide
expression pattern of Pax6-positive, lens-biased tissue, additional
signaling is clearly required to repress lens fate in most of the PPR
and maintain it only in the presumptive lens. Sullivan et al. (2004)
suggested that neural crest cells, which begin migrating within the
head at HH stage 9 of chick development (7 somites), repress lens
fate in all areas apart from the presumptive LE, which is connected
to the OV and is thus protected from neural-crest repression. Neural
crest cells were shown to repress lens development in the PPR in
explants, and when they were ablated in the chick, ectopic lenses
formed in the non-ocular ectoderm (Bailey et al., 2006).
Several paracrine signals have been suggested to inhibit lens
fate in the head ectoderm. Most notably, the canonical Wnt
pathway has been shown to be restrictive to lens development.
Deletion of b-catenin, the downstream effector of canonical Wnt
signaling, results in the appearance of multiple crystallinexpressing lentoid bodies around the normal lens (Smith et al.,
2005). Accordingly, overexpression of b-catenin in the lens
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primordium downregulates Pax6 expression and completely
prevents LP formation (Kreslova et al., 2007; Smith et al., 2005).
TGFb signaling was recently shown to be a mediator of neural
crest repression of lens fate. TGFb proteins and receptors are
expressed in the ocular primordium, and functional studies have
revealed their importance in tissue interactions in the developing
eye. Grocott et al. (2011) demonstrated that neural crest cells
secrete TGFb, which can induce Smad3 activity in the periocular
ectoderm. Active Smad3 is itself capable of preventing Pax6 activity
in the SE, through direct binding to the protein and prevention of
Pax6’s auto-regulation (Grocott et al., 2007). Moreover, the authors
showed that TGFb signaling activates Wnt2b expression in the
ectoderm, and the neural crest may therefore inhibit lens fate
indirectly by causing the ectoderm to activate canonical Wnt
signaling (Grocott et al., 2011).
In concert with Wnt inhibition of Pax6 in the surrounding
ectoderm, Pax6 in the LP itself counteracts this inhibition by activating anti-Wnt genes. Pax6 has been shown to directly activate the
Wnt antagonists Sfrp1, Sfrp2, and Dkk1 (Machon et al., 2010). In
addition, some ectopic activation of canonical Wnt signaling was
observed in the epithelium of Pax6sey/sey despite contact with the
OV. Therefore, Pax6 acts in several ways to ensure LP formation in
the area of contact with the OV.
3.3.2.2. The role of the OV in lens induction. Initiation of morphogenesis of the LP from the prospective lens-forming ectoderm
occurs after its contact with the underlying OV. Studies have
implicated several signaling pathways in LP induction: FGF (Faber
et al., 2001; Garcia et al., 2011; Gotoh et al., 2004; Nakayama
et al., 2008; Pan et al., 2006; Vogel-Hopker et al., 2000), Notch
(Ogino et al., 2008) and BMP (Furuta and Hogan, 1998; Pandit et al.,
2011; Rajagopal et al., 2009; Sjodal et al., 2007; Wawersik et al.,
1999; Yang et al., 2010).
The role of FGF signaling in lens induction is still under debate.
Several mouse genetic models support the necessity of FGF
signaling in this process, as evident from arrested LP formation and
down-regulation of Pax6. These models include a truncated dominant-negative receptor (Fgfr1IIIc), mutations in the FGF-receptoradapter Frs2a, and a germline deletion of Ndst1, which is required
for the biosynthesis of heparan sulfate proteoglycans (Faber et al.,
2001; Gotoh et al., 2004; Pan et al., 2006). In contrast, mutations
in several FGF ligands did not prevent LP formation. This could be
attributed to redundancy among several FGF ligands that are
expressed in the eye (Smith et al., 2010). To directly test the roles of
FGFs in lens induction, Garcia et al. (2011) deleted both FgfR1 and
FgfR2 receptor genes from the lens pre-placode using conditional
alleles and the Le-Cre transgene. Surprisingly, expression of Pax6,
Six3 and other placodal markers was maintained in the double
mutants, while increased apoptosis was detected in the mutated LP
(Garcia et al., 2011). These findings led to the conclusion that the
main role of FGF signaling in mouse is not in lens induction, but
rather in survival of cells of the lens primordium (Garcia et al.,
2011).
A role for the Notch cell-cell signaling pathway has been
implicated in lens induction (Lai, 2004). A recent study conducted
in Xenopus embryos suggests that Notch signaling mediates cross
talk between the OV and the overlying SE (Ogino et al., 2008). In
Xenopus embryos, the receptor Notch2 is found in the pre-placode
while the ligand Delta2 is highly expressed in the OV (Ogino et al.,
2008). Functional binding sites for Otx2 and RbpJk were identified
within an enhancer sequence of FoxE3, which is expressed in the LP
and is required for cell proliferation and detachment of the lens
vesicle from the SE (Blixt et al., 2000; Brownell et al., 2000). This
suggests that the Notch ligands in the OV function to activate Notch
within the pre-placodal ectoderm and that this signaling, combined
15
with intrinsic Otx2, regulates FoxE3 (Ogino et al., 2008). The role of
Notch signaling in lens induction has yet to be demonstrated in
mammals.
BMP signaling has been found to be essential for LP formation.
Germline mutations in Bmp4 as well as Bmp7 prevent formation of
the LP (Dudley et al., 1995; Furuta and Hogan, 1998; Jena et al., 1997;
Luo et al., 1995; Wawersik et al., 1999). Bmp7 is expressed in the SE
and surrounding mesenchyme and is required for maintenance of
Pax6 and Sox2 expression in the SE (Wawersik et al., 1999). In
contrast, Bmp4 is strongly expressed in the OV and is therefore
a good candidate for the execution of OV-mediated lens induction.
Accordingly, tissue recombination and rescue experiments on
a Bmp4-null background revealed that it is required for the lensinducing activity of the OV (Furuta and Hogan, 1998). Finally,
somatic inactivation of the BMP receptor genes Bmpr1a and Acvr1,
as well as the Smad-encoding genes 1, 4 and 5, exclusively in the SE
abrogated lens induction and revealed distinct roles for each
receptor (Rajagopal et al., 2009). Bmp4 functions in parallel to Pax6
in lens induction as it is expressed in Pax6 mutants and Pax6 is
detected in Bmp4 mutants (Furuta and Hogan, 1998). Sox2 seems to
be a target of both Bmp4 and Pax6 as its expression is lost in Bmp4null embryos and Pax6 SE somatic mutants (Ap2a-cre; Pax6loxP/loxP)
(Furuta and Hogan, 1998; Smith et al., 2009). As Sox2 seems to be
dispensable for LP formation in the mouse (see section 3.2.3), it is
expected that additional Bmp4 and Pax6 targets are involved in
mediating its activity in LP formation (Smith et al., 2009).
As the eye field and OV act as inducers of lens development, an
open question remains as to what is the effect of EFTFs in the OV on
the signaling cascade that results in the activation of genes in the
SE. Lhx2 in the OV was found to regulate both Bmp4 and Bmp7 (Yun
et al., 2009). The reduced expression of BMPs could, however, be
indirect, due to the altered morphology and specification of the OV
in Lhx2 mutants (Yun et al., 2009). To resolve the function of eyefield genes, including Pax6, in the process of lens induction,
future studies should employ temporally regulated transgenic Cre
lines that target genes exclusively in the OV.
3.3.3. Pax6 is required in a stage-dependent manner in the
transition of PPR to lens placode
The roles of Pax6 prior to and during formation of the LP were
initially investigated through recombination assays of mouse and
rat germline mutants, as well as studies of chimeric embryos
composed of combined wild-type and Pax6-deficient cells
(Collinson et al., 2001; Fujiwara et al., 1994; Quinn et al., 1996).
These studies pointed to an autonomous role for Pax6 in the SE
during LP formation. The direct proof for Pax6 requirement in the
SE was obtained through a series of somatic mutations in the SE,
which also allowed for the discovery of genetic pathways downstream of Pax6. Three somatic mutants of Pax6 in the SE were
established, each targeting Pax6 in distinct stages of lens induction
(Fig. 2). Loss of Pax6 at the PPR (E8.5) was achieved using the Ap2acre;Pax6loxP/loxP somatic mutant (Macatee et al., 2003; Smith et al.,
2009). Slightly later inactivation in the pre-placode LE (E9) was
observed using the Le-cre;Pax6LacZ/loxP, which carries one null allele
(St-Onge et al., 1997) and one conditional allele of Pax6 (AsheryPadan et al., 2000; St-Onge et al., 1997). Finally, in the Le-cre;Pax6loxP/loxP, Pax6 was deleted at the LP stage on around E9.5 (Fig. 2)
(Ashery-Padan et al., 2000; Huang et al., 2011; Smith et al., 2009). In
this series of experiments, Pax6 was found to be essential for the
lens-forming competence of the PPR. In Pax6-null mutants,
expressions of Sox2, Sfrp2 and Six3 were not detected in the prelens placode. Complete blockage of lens development was also
observed following Cre/loxP mutation of Pax6 exclusively in the LE
of an Ap2a-cre;Pax6loxP/loxP somatic mutant (Smith et al., 2009). The
arrest in the onset of lens-specific genes in both the germline and
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O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
somatic SE Pax6 mutants unequivocally demonstrated a cellautonomous role for Pax6 in the PPR in the initiation of a genetic
program controlling lens formation.
The conditional mutant series further revealed that Pax6 plays
a stage-dependent role in the dynamic events taking place prior to
and during morphogenesis of the LP. This is best exemplified
through analysis of the regulatory and cooperative interaction
between Pax6 and Sox2 (Kamachi et al., 2001; Smith et al., 2009).
Several studies have inferred an important role for Sox2 during the
early stages of lens development. In the chick, Sox2 was found to
regulate d1-crystallin cooperatively with Pax6, as well as its own
expression (Inoue et al., 2007; Kamachi et al., 2001). Furthermore,
Sox2 was shown to be required for the expression of N-cadherin
(CDH2), an adhesion molecule that is required for lens morphogenesis (Pontoriero et al., 2009). Sox2 is elevated in the PPR in
response to inductive cues from the OV (see section 3.3.2.2)
(Kamachi et al., 1998). Somatic mutations of Sox2 in the pre-placode
and placode stages (Ap2a-cre;Sox2loxP/loxP and Le-cre;Sox2loxP/loxP,
respectively) revealed that Sox2 is dispensable for LP formation but
is required for subsequent stages of lens morphogenesis and
differentiation (Smith et al., 2009).
To further explore the epistatic relationship and function of Pax6
and Sox2, a conditional mutant in one of the genes was compared
with the double somatic mutants. Sox2 expression was retained in
the SE of the Le-cre;Pax6LacZ/loxP; however, when Pax6 loss was at
the LP stage, Sox2 expression was reduced (Smith et al., 2009). This
suggests that early on, Pax6 and Sox2 function in parallel, whereas
Pax6 is required for Sox2 expression in the placode itself. Further
analysis revealed that Sox2 is required independently of Pax6 for
onset of N-cadherin at the pre-placode stage. In the LP, Sox2 as well
as N-cadherin are dependent on Pax6 activity (Smith et al., 2009;
van Raamsdonk and Tilghman, 2000). As cadherins play a vital
role in placode formation and separation of the lens pit from the
ectoderm (Pontoriero et al., 2009), Pax6 may be quantitatively
required for N-cadherin-mediated selection of cells from the placode in the detachment process.
The above findings reveal Pax6’s involvement in several steps of
lens induction, starting with conferring lens competence in the PPR
and ending with the ability of the pre-placode to activate downstream genes that are required during further stages of lens
development.
3.3.4. Morphogenesis of the lens placode and lens pit
Sensory placodes are characterized as thickenings of columnar
ectoderm, which in the lens is followed by the invagination required
to form the lens vesicle. Prior to and during placode formation and
invagination, ECM accumulates between the LP and OV (Hendrix
and Zwaan, 1975). Placode thickening is hypothesized to take
place via anchoring of the epithelium to factors in the ECM in
concert with proliferation. These two processes result in “crowding”
of the cuboidal cells of the epithelium and create the necessary force
for elongation into columnar cells (Hendrix and Zwaan, 1974).
Support for this hypothesis comes from a recent study in which the
ECM component Fibronectin1 (Fbn1) was deleted from the eye
primordium. In this mutant, the LP was induced, as reflected by
gene expression and cytoskeletal rearrangement, but the placode
did not thicken or invaginate (Huang et al., 2011).
Similar to the phenotype of the Fbn1 mutants, cell crowding
does not occur in the SE of the Le-cre;Pax6loxP/loxP somatic mutants.
Instead, the area of the ectoderm that is in contact with the OV
spreads laterally. Corresponding with this morphogenic phenotype,
reduced expression of ECM molecules such as Fbn1 was observed in
the Pax6-deficient SE (Huang et al., 2011). These findings infer a role
for Pax6 in the regulation of ECM molecules which, in turn, are
essential for the physical formation of the LP (Huang et al., 2011).
Invagination of a planar tissue such as the LP requires
constriction of the apical side of the epithelium while the basal side
remains anchored to the basal lamina (Hendrix and Zwaan, 1974).
An actin-binding protein, Shroom3, was shown to enable apical
constriction in the lens by recruiting F-actin, Myosin2, and Vasp to
the apical side of the placode (Plageman et al., 2010). In Le-cre;Pax6loxP/loxP mutants, Shroom3 expression is lost, while misexpression of Pax6 in frog embryos induces Shroom3 (Plageman
et al., 2010).
Therefore, in addition to activating transcription factors of the
lens lineage, Pax6 is also important for regulating genes that
function in determining cell shape and tissue morphogenesis.
3.3.5. A diploid level of Pax6 is required for detachment of the lens
vesicle from the surface ectoderm
The next step of lens development requires “pinching off” of
the invaginating LP (the “lens pit”) in order to form a detached
lens vesicle. While a complete Pax6-null model of this stage is
unavailable, lens vesicle detachment seems to be the stage most
sensitive to the level of Pax6. In both aniridia patients and the
mouse heterozygous Small eye model (Pax6Sey/þ), the lens fails to
detach from the overlying cornea. This haploinsufficiency of Pax6
is intrinsic to the cells of the lens lineage, as conditional knockout
of one copy of Pax6 specifically in the lens primordium recapitulates the heterozygous Pax6Sey/þphenotype (Davis et al., 2009). The
failure in lens vesicle detachment in Pax6Sey/þ could be attributed
to the reduced size of the LP due to a developmental delay (van
Raamsdonk and Tilghman, 2000). An additional explanation is
that the transcription of a downstream target of Pax6 at this stage
is extremely sensitive to Pax6 protein levels. The transcription
factor FoxE3 is required for proliferation and survival of the lens
epithelium and for detachment of the lens vesicle (Blixt et al.,
2000; Brownell et al., 2000). Two other transcription factors,
Sip1 (ZEB2) (Yoshimoto et al., 2005) and Mab21l (Yamada et al.,
2003), were found to be upstream regulators of FoxE3. As
neither Mab21l1 nor FoxE3 are detected in Pax6-null mutants, it
has been suggested that Pax6 regulates both genes during early
stages of lens development (Yamada et al., 2003). More importantly, FoxE3 expression is almost completely lost in the lens pit of
Pax6Sey/þ heterozygotes (Blixt et al., 2000). At later stages, after
lens morphogenesis is complete, Pax6 is not required to maintain
FoxE3 expression in the lens epithelium (Shaham et al., 2009).
Therefore it is highly plausible that haploid levels of Pax6 are
insufficient for the transcription of FoxE3 (either directly or
through Mab21l) at the time of lens vesicle detachment, which is
in turn responsible for the persistent lens stalk in Small eye mice
and in human aniridia patients.
The findings presented in sections 3.3.4 and 3.3.5 place Pax6
upstream of molecules that control cell shape and tissue
morphology (i.e. Fbn1, Shroom3 and N-cadherin) and developmental regulators that function to control cell proliferation and
survival (i.e. Sox2, FoxE3, Mab21l1). Apart from Sox2, it is unclear
whether other Pax6 genetic targets are regulated directly or indirectly by Pax6 during lens morphogenesis. Studies of mutants in
which Pax6 and other genes are inactivated in the lens pit stage are
needed to determine the gene network required for lens
morphogenesis.
Reviewing the different Pax6 phenotypes during lens morphogenesis, it appears that after placode formation, Pax6 level determines the cells’ ability to exit the SE and contribute to the proper
lens lineage. Growth, invagination and detachment of the lens pit
all happen in concert through the activity of several genes implicated as downstream targets of Pax6. Further insight into this
developmental process will contribute to our understanding of the
cellular events required for epithelial morphogenesis.
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3.4. Roles of Pax6 in the transition from lens progenitors to
differentiated cells
3.4.1. Lens fiber differentiation
Following detachment from the SE, the lens vesicle acquires
a polarized morphology. The anterior cells retain their epithelial
and proliferative properties while the posterior lens cells exit the
cell cycle, and differentiate into primary lens fiber cells (LFCs) that
elongate and fill the lumen of the vesicle. From this stage onward,
new secondary lens fibers are continuously added at the lens
equator (from around E12.5 in mouse; Kallifatidis et al., 2011).
During secondary LFC differentiation, cells at the equator undergo
their terminal mitosis, move posteriorly to the transition zone,
acquire fiber morphology and accumulate an array of crystallin
proteins that are required for lens transparency and light refraction
(Bassnett and Beebe, 1992; Beebe et al., 1982; Piatigorsky, 1981;
Rafferty and Rafferty, 1981). At late stages of mouse embryogenesis, the fibers at the center of the lens lose their organelles,
including their nuclei. Organelle loss is essential for lens transparency and normal vision (Kuwabara and Imaizumi, 1974;
Nishimoto et al., 2003). The acquisition of new LFCs through LE
differentiation and maturation continues throughout mammalian
17
life. The new lens fibers are deposited at the lens equator,
producing an onion-like lens structure with the oldest, mature LFCs
in the center of the lens and younger LFCs toward the periphery.
Analyses of the expression of genes involved in cell proliferation
and differentiation reveal the dynamic events occurring during the
transition of the lens epithelium to differentiated fibers. During the
period of rapid embryonic growth, extensive proliferation occurs
throughout the lens epithelium. At the transition zone (Fig. 5A, B),
these progenitors differentiate into new lens fibers and contribute to
tissue growth through both an increase in cell number and a constant
increase in the length of the new cell fibers (Kallifatidis et al., 2011).
As the rate of lens growth declines, the proliferation and differentiation of new fibers is reduced. Close to birth and in postnatal
development, proliferation is mostly restricted to the lens periphery,
termed the germinal zone, while a few slow-cycling cells are
detected in the anterior lens epithelium. These regions are considered to contain the lens stem cells in the adult (Zhou et al., 2006).
During the rapid growth phase of the lens in the embryo, there
are clear molecular distinctions between the lens epithelium and
the transition zone (Fig. 5B). The lens epithelium is characterized by
expression of the transcription factors Ap2a, FoxE3 and the celladhesion molecule E-cadherin. These genes are downregulated in
Fig. 5. Molecular pathways and Pax6 regulatory network during secondary lens fiber cell (LFC) differentiation. (A) Illustration of a segment of the mouse lens during secondary LFC
differentiation (E14.5). Colored patterns mark cells at different stages of differentiation. (B) The table shows expression of representative genes in the various compartments. Color
gradient of the bars represents level of expression. (C) Histology (Hematoxylin and Eosin staining) of the control and Pax6loxP/loxP;Mrl-10Cre E14.5 lens. (D) A proposed genetic
network in secondary LFC differentiation. Pax6 is required for survival of the anterior lens epithelium, cell-cycle withdrawal and differentiation of transition-zone lens epithelium to
LFC. Other transcription factors are independently required for different stages of differentiation, while elevation of Wnt/b-catenin inhibits differentiation. Pax6 activates Wntantagonists, such as Sfrp2, which may alleviate Wnt inhibition.
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
(2012), doi:10.1016/j.preteyeres.2012.04.002
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O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
the transition zone where cells are not dividing (Fig. 5). Corresponding with cell-cycle exit, cyclin-dependent kinase inhibitors
Cdkn1b (p27KIP1) and Cdkn1c (p57KIP2) and LFC-differentiation
transcription factors (i.e. Sox1 and c-Maf) are upregulated in the
transition zone (Kamachi et al., 1998; Kawauchi et al., 1999). In
addition, Prox1 protein, which is localized to the cytoplasm of the
lens epithelia, accumulates in the nuclei in the transition zone,
where it is required for regulation of cell-cycle inhibitors (Duncan
et al., 2002; Wigle et al., 1999).
The distinction between anterior and peripheral lens epithelium
is less apparent than that with the transition zone, as proliferation
occurs in both regions. There are, however, recent reports on genes
that are expressed with distinct patterns within the LE, indicating
early partitioning to central anterior vs. peripheral epithelial
domains. Notably, the Notch signaling pathway seems to function
at the border between the transition zone and the lens epithelium
(Jia et al., 2007; Le et al., 2009; Rowan et al., 2008; Saravanamuthu
et al., 2009). The Notch ligand Jagged1 (Jag1), which is required for
lens epithelium proliferation and differentiation, is detected at low
levels in the peripheral epithelium and at higher levels in the
transition zone (Le et al., 2009). Jag1 is thought to trigger Notch
juxtacrine signaling back to the lens epithelium, and the cells of the
peripheral lens epithelium are exposed to more Jag1 ligands due to
the shape of the transition zone (Fig. 5, Le et al., 2009;
Saravanamuthu et al., 2009). This distribution of Jag1 may result
in elevated activity of Notch, as evidenced by the expression of Hes5
in the peripheral lens epithelium (Saravanamuthu et al., 2009; and
these authors’ unpublished observations).
3.4.2. Pax6 is required for differentiation of secondary lens fibers
In contrast to the restricted expression of the above genes, Pax6
is detected in the anterior and peripheral lens epithelium as well as
in the transition zone. The expression of Pax6 is reduced in young
fiber cells and lost in the differentiated fibers. This broad pattern of
expression alludes to different roles for Pax6 in each of the lens
compartments. Recently, using somatic mutation with a lensspecific Cre line, we examined the role of Pax6 during the later
stages of lens development. Somatic mutation of Pax6 from the lens
was performed using the Mlr10Cre transgene which is active in the
lens exclusively during secondary fiber differentiation (Shaham
et al., 2009; Zhao et al., 2004). This conditional mutation of Pax6
(Pax6loxPloxP;Mlr10Cre) resulted in failure of the lens epithelial cells
to exit the cell cycle, arrested LFC differentiation and elevated
apoptosis (Shaham et al., 2009). Although Pax6-deficient anterior
lens epithelial cells eventually die, those that were retained did not
lose their lens epithelium phenotype as the expression of aA and aB
crystallin was detected in the anterior lens epithelium. This indicates that Pax6 is dispensable at this stage for maintaining anterior
lens epithelium identity. Alternatively, the mRNA and proteins
produced prior to Pax6 mutation are sufficient to maintain the
anterior lens epithelium phenotype until these cells are lost due to
apoptosis.
In addition to the function of Pax6 in cell survival in the Pax6deficient lens, a major phenotype of the Pax6 mutant was
observed in the transition zone. cMAF, Sox1 and nuclear Prox1 were
detected at the equator of the Pax6loxPloxP;Mrl10Cre lenses, suggesting that the onset of differentiation in the transition zone had
taken place. However, BrdU and Ki67þ cells were detected in the
transition zone as well as posterior to the lens equator (Shaham
et al., 2009). Thus, onset of lens differentiation can occur in Pax6deficient lenses but the cells fail to exit the cell cycle, elongate as
fiber cells and accumulate b- and g-crystallin. Therefore, Pax6 is
specifically required for lens fiber differentiation at the transition
zone. The failure to initiate fiber crystallins is surprising considering the inhibitory role of Pax6 on these genes documented in cell
culture (Duncan et al., 1998; Kralova et al., 2002). The outcome of
Pax6 loss in vivo infers multiple events that must occur simultaneously to allow differentiation of the LFCs. The study of Pax6’s role
during secondary fiber differentiation exemplifies the complexity
of this gene, even in a seemingly simple structure such as the lens.
Pax6 has distinct functions in each of the lens compartments. Thus,
elucidating the genetic pathways downstream of Pax6 must take
into account multiple networks regulating the cell cycle, onset of
differentiation, cell morphology and survival.
The role of Pax6 in LFC differentiation may also be related to
mediating external responses. The cells of the lens respond to
multiple growth factors in the surrounding media (reviewed in
Lovicu et al., 2011). FGF signaling seems to be the most promising
candidate in this regard, as it is pivotal for the differentiation of
LFCs in vivo and in vitro (Zhao et al., 2008).
Another extensively researched pathway in this context is the
Wnt pathway. Recent studies have documented inhibitory regulation between Pax6 and Wnt signaling during early stages of lens
development (see section 3.3.2.1). Sfrp2 is dependent on Pax6
activity during the stages of secondary fiber differentiation as well
(Shaham et al., 2009). Moreover, overactivation of the pathway
prevents LFC differentiation (Martinez and de Iongh, 2010; Shaham
et al., 2009). Even so, based on transgenic reporters, canonical Wnt
signaling is absent from the lens during later stages of development
(Liu et al., 2003, 2006a, 2007; Shaham et al., 2009). Removal of Pax6
and Sfrp2 from the lens did not result in activation of canonical Wnt
signaling in the Pax6-deficient lenses (Shaham et al., 2009). This
suggests that there are additional, Pax6-independent mechanisms
that function to inhibit b-catenin transcriptional activity in the lens.
It is also possible that Pax6 is involved in regulation of the noncanonical Wnt planar cell polarity pathway (Wnt/PCP), as Sfrp2
has been shown to disrupt Wnt/PCP signaling in the lens (Sugiyama
et al., 2010).
3.4.3. Pax6 down-regulation is required at the final stages of lens
differentiation
Within the lens cortex, secondary LFCs begin their final differentiation, which is characterized by elongation and accumulation
of fiber-specific crystallins of the bg-superfamily, as well as several
intermediate cytoskeletal fibers, such as beaded filaments (Song
et al., 2009). As fiber cells mature, they also lose most of their
organelles, including the nucleus. Final lens-fiber differentiation
and maturation is accompanied by an obvious downregulation of
Pax6. It is therefore difficult to ascertain the role of Pax6 in these
later processes. However, actual removal of Pax6 from the mature
LFC seems to be important for the differentiation process. In ectopic
gain-of-function experiments, activation of Pax6 in fiber cells
resulted in reduction in c-Maf, reduced accumulation of lens crystallins and failure of fiber cells to form interlocking structural digitations (Duncan et al., 2004). Therefore, while Pax6 is required for
the onset of LFC differentiation, eventual reduction of the protein is
required for the final stages of LFC maturation.
3.5. Roles of Pax6 in corneal development and homeostasis
The adult cornea consists of three major layers that originate
from two embryonic cell types: the stratified surface of corneal
epithelial cells is derived from the surface ectoderm, while the
keratocytes of the corneal stroma and the inner endothelial layer
both originate from periocular mesenchyme (Trainor and Tam,
1995). In the mouse, corneal maturation occurs after birth as the
two layers of epithelium become a self-renewing stratified
epithelium and the innervation and vascular processes take place
(reviewed in Cvekl and Tamm, 2004). The limbus, the junction
between the cornea and conjunctiva, is important for the renewal
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
(2012), doi:10.1016/j.preteyeres.2012.04.002
O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
of the corneal epithelium in the adult as it contains limbal epithelial
stem cells (Secker and Daniels, 2008).
Pax6 is expressed in the SE prior to and during corneal differentiation and is maintained in the adult epithelium, including the
limbus. Analysis of Pax6þ/þ 4 Pax6/ chimeras revealed that Pax6
is required autonomously in SE cells for these to be incorporated
into the corneal epithelium (Collinson et al., 2003). In addition to its
requirement for initial specification of the corneal lineage, development of the cornea requires an exact diploid dosage of Pax6, as
both reduction and elevation in Pax6 abrogate corneal development (Davis and Piatigorsky, 2011; Dora et al., 2008; Mort et al.,
2011; Ramaesh et al., 2003; Schedl et al., 1996). Notably, a significant component of the vision loss in aniridia patients is due to the
corneal abnormalities collectively known as aniridia-related keratopathy (ARK). ARK in humans and in Pax6þ/ mice includes
peripheral vascularization, opacification of the stroma, thin and
irregular corneal epithelium with ectopic goblet cells, conjunctivalization and abnormal corneal innervation (Leiper et al.,
2009; Ramaesh et al., 2003).
Initially, ARK was presumed to be due to malfunction of the
limbal stem cells resulting in invasion of conjunctival cells into the
cornea proper and failed wound repair (Nishida et al., 1995).
However, several additional explanations for the involvement of
Pax6 in ARK have been offered in the last few years. First, in Pax6þ/
mice, neural crest cells are abnormally distributed in the eye
including the corneal stroma and endothelium. Therefore, a correct
dose of Pax6 is required in a non-autonomous mechanism for the
proper recruitment of keratocytes from the neural crest (Kanakubo
et al., 2006). It is possible that reduced expression of retinoic acid in
Pax6 mutants accounts for this migratory defect (Duester, 2009;
Suzuki et al., 2000). In addition, Pax6þ/ mice and human aniridia
patients are deficient in a secreted form of VEGF receptor 1 (SFLT-1),
which is normally expressed in epithelial cells and inhibits vascularization of the corneal stroma. Reintroduction of this protein into
Pax6þ/ mice restores corneal avascularity (Ambati et al., 2006).
However, how Pax6 controls the expression or secretion of this
mediator of corneal opacity is still unknown.
Pax6 dosage may also be intrinsically important in the epithelial
compartment. The fragility of the epithelium in Pax6þ/ corneas
was associated with deficient expression of cytokeratins in the
epithelial cells themselves. Cytokeratin proteins are in turn essential for the integrity of the corneal epithelium (Davis et al., 2003;
Kao et al., 1996; Ou et al., 2010). Pax6þ/ epithelial cells have
abnormal cell junctions and cytoskeletal organization, which result
in a chronic wound-healing defect that contributes to the ARK
phenotype (Ou et al., 2010).
Considering the above, it is evident that Pax6 plays multiple
roles in corneal development and function. Further understanding
of the etiology of ARK and the involvement Pax6 in corneal
development, homeostasis and wound healing will require
comprehensive analyses of somatic Pax6 mutations in the limbal
and epithelial compartments of the cornea.
4. Conclusions and future directions
The extensive research on Pax6 activity at multiple levels in the
last few decades has amassed a great deal of data on its role in
acquisition of cell fate and homeostasis of differentiated cell types.
Notwithstanding, we remain astounded by its capacity to execute
multiple activities. Pax6 functions upstream of lineage regulators
that trigger distinct developmental programs, such as Mitf during
RPE specification and bHLH factors in the specification of retinal
cell types. In addition, some downstream targets of Pax6 are
directly required for cellular morphology, adhesion and migration,
19
while other targets are required for cellular homeostasis and
survival.
Major advances in our understanding of the Pax6 gene network
have been made through an unbiased examination of transcriptome changes in Pax6 mutants, utilizing microarrays and other
high-throughput technologies, as well as identification of Pax6binding regions using chromatin immunoprecipitation. To date,
changes in gene-expression profiles have been determined in Pax6
heterozygous and gain-of-function lenses (E15, P0) (Chauhan et al.,
2002a,b; Wolf et al., 2009), Pax6-deficient SE (Huang et al., 2011),
and Pax6-deficient and overexpressing neuronal tissues (Arai et al.,
2005; Duparc et al., 2006; Holm et al., 2007; Numayama-Tsuruta
et al., 2010; Sansom et al., 2009; Shimizu et al., 2009; Visel et al.,
2007). On-going studies of defined developmental stages and tissue
types are expected to contribute to further expanding the known
pool of Pax6 targets. In addition to determining the genetic
network mediating Pax6’s spatiotemporal activity, biochemical and
transgenic approaches must be combined with in silico analyses to
resolve how Pax6 binding to DNA sequences mediates quantitative
regulation of gene expression during embryogenesis.
To uncover the molecular mechanism of Pax6 activity in vivo, the
combined effects of other transcription factors and cofactors
operating in the specific tissue must be taken into account. This is
exemplified by recent studies on the sequence selectivity of the Hox
family of homeodomain transcription factors (reviewed in Ansari
and Peterson-Kaufman, 2011). In a study by Slattery et al. (2011),
SELEX-seq was applied to determine sequences bound by heterodimers of Hox proteins and the Extradenticale cofactor (exd). This
approach revealed novel binding sites for the heteromeric complex,
which account at least in part for the changing specificity of Hox
proteins during animal development. Pax6 target selection must be
similarly dependent on its interacting partners. Sox2 is one of
several interacting partners that function with Pax6 during early
stages of lens development. Employing an in-vitro systematic
approach to identify the binding sites of the heterodimeric complex
as well as ChIP-seq of both proteins in lens cells could lead to an
understanding of their target specificity during early stages of lens
development, and shed light on the mechanisms that mediate
highly specific functions of developmental transcription factors
during organogenesis.
In addition to cooperation with lineage-specific transcription
factors, a promising mechanism that might explain the various
functions of Pax6 is its ability to interact with general chromatin
modifiers, which alter the state of chromatin in regulatory regions.
As mentioned above, Pax6 interacts with the chromatin modifiers
Brg1 and p300 to modulate lens crystallin expression (Cvekl and
Duncan, 2007; Cvekl and Mitton, 2010). Further research is
needed to understand how chromatin remodeling affects promoter
accessibility and how gradual changes in chromatin state enable
orchestrating
multiple
differentiation
programs
during
organogenesis.
Another interaction that controls the outcome of Pax6 activity is
the presence of different growth factors and activation of signaling
pathways. Signaling pathways are reused multiple times during
development, while organ-specific regulators such as Pax6 determine the particular response to the positional information. To
elucidate the mechanism underlying differences in Pax6’s activity
within different tissue types it is therefore required to map the
convergence of the extracellular information available to the tissue,
and to determine how this interaction differs in different locations.
The extrinsic cues can also trigger biochemical modification of the
Pax6 protein, affecting its stability or transcriptional properties,
essentially making it part of the signaling cascade. An example of
this is the TGFb-mediated inhibition of Pax6 in the SE (see section
3.3.2.1). An additional intersection for extrinsic and intrinsic cues is
Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
(2012), doi:10.1016/j.preteyeres.2012.04.002
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O. Shaham et al. / Progress in Retinal and Eye Research xxx (2012) 1e26
the interaction of Pax6 and transcription factor effectors of
signaling pathways on enhancers of target genes. This paradigm
has been reported for the mediation of tissue-specific activity of
master regulatory genes in the fly thorax and further implicated for
retinal determination genes in the fly (Baker and Firth, 2011;
Curtiss et al., 2002; Mann and Carroll, 2002).
As we showed in this review, Pax6 is maintained in specific cell
types of the adult CNS and eye, in addition to its activity in the
embryo. However, knowledge on its functions in differentiated cell
types is lacking. Recently, Pax6 was found to play a crucial role in the
survival of adult dopaminergic cells in the olfactory bulb, implicating a neuroprotective activity (Ninkovic et al., 2010). In the adult,
Pax6 is further detected in myriad stem cells and has been found to
be required for adult neurogenesis in the hippocampal sub-granular
zone and in the sub-ventricular zone of the neocortex (Hack et al.,
2005; Maekawa et al., 2005). In the eye, Pax6 is maintained in
several cell types that are implicated to maintain self-renewal
properties and in regeneration of ocular structures in several
animal species: in the lens epithelium, the corneal limbus, the pigmented CB and iris, and at low levels in the Muller glia (reviewed in
Karl and Reh, 2010). It would be of interest to understand Pax6
activity in these adult cell types and to determine if there are
common targets that mediate activities relating to embryonic
development, adult self-renewal and organ regeneration.
Collectively, the abundance of research performed on this
fascinating gene has led to advances in dogmas of developmental
biology such as tissue induction and stepwise differentiation.
Furthermore, the understanding of Pax6 activity during development and tissue maintenance will lead to a better understanding of
diseases that are associated with Pax6 reduction or altered
expression including glaucoma, retinal degeneration, cataracts and
corneal diseases. A further understanding of the regulation of Pax6,
the modulation of its expression levels and its dosage-dependent
activity during development and in the adult cell types will shed
light on etiology of ocular diseases and provide an important step in
designing cellular and molecular therapies.
Acknowledgments
We thank Varda Wexler for assisting with the graphic design.
R.A-P.’s research is supported by the Israel Science Foundation
(grant number 610/10), Morasha foundation (1372/11), the Israel
Ministry of Science (grant number 36494), the Ziegler Foundation,
the Binational Science Foundation (grant number 2007243), the
German Israeli Foundation (grant number 1128-156.1/2010), the
Israel Ministry of Health (grant number 3-7439), Maratie foundation, and the IsraeleItaly joint innovation program.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.preteyeres.2012.04.002.
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Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
(2012), doi:10.1016/j.preteyeres.2012.04.002
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Please cite this article in press as: Shaham, O., et al., Pax6: A multi-level regulator of ocular development, Progress in Retinal and Eye Research
(2012), doi:10.1016/j.preteyeres.2012.04.002