Issue 1, December 1998
A Review of the Highly Conserved PAX6 Gene in Eye Development
Adam A. L. Friedman
Aniridia is a human genetic disease that is manifested by alterations
in the structure and function of the eye, including reduced iris
size, absence of the fovea, and lens deformities (Glaser, et al.
1992). First documented as a genetic disease over 150 years ago,
it has since become a model for autosomal dominant genetic disorders
because of the high penetrance of its mutant alleles, the ease of
diagnosis at birth, and a similar incidence in various populations
(Glaser, et al. 1995). It was not until recently, however, that
the aniridia gene (AN) was mapped (to chromosome band 11p13) and
determined to be the gene PAX6 (Glaser, et al. 1992), a regulator
of development of the eyes and central nervous system. There is
a PAX6 dosage effect in aniridia ranging from mild loss of visual
acuity and cataracts to severe nervous system defects and
anopthalmia (complete absence of the eyes) (Glaser, et al. 1994).
Many of the studies that led to the identification of PAX6 as the
human aniridia gene were conducted in mammals and insects and demonstrated
that PAX6 is highly conserved among vertebrates and lower animals.
PAX6 homologues have been found in mice (Small eye, or Sey), rats,
zebrafish, quail, and the fly Drosophila (eyeless, or ey) with amino
acid sequence identities of approximately 90% (Quiring, et al. 1994);
PAX6 is 96% identical in its amino acid sequence to the pax(zf-a)
in the zebrafish, but the two species diverged over 400 million
years ago (Glaser, et al. 1992). This degree of conservation approaches
that of histones, some of the most highly conserved proteins known.
More important, however, is the finding that Drosophila and vertebrates
have homologous PAX6 genes with similar functions and similar mutant
phenotypes. In Origin of the Species, Darwin found it difficult
to explain the evolution of structures as dissimilar as simple vertebrae
eyes and compound insect eyes; he speculated that the structures
might have developed separately through convergent evolution (Zuker,
1994). However, in both insects and vertebrates, PAX6 is
expressed in the embryo just prior to and during formation of the
eye in the region of its development. Based on that evidence, and
the finding that misexpression of a PAX6 homologue in flies
could induce ectopic eyes, it has been suggested that PAX6
is a master regulatory gene that induces eye development in a broad
range of animals (Halder, et al. 1995).
The PAX multigene
The PAX6 protein is one of many transcription factors that induce
embryonic differentiation along the major body axes. In response
to concentration gradients of other regulatory proteins, these transcription
factors bind to specific DNA sequences of other genes and regulate
their expression, thus translating positional information into developmental
patterns for distinct structures (Glaser, et al. 1994).
PAX6 is a member of the PAX multigene family of transcription
factors that help regulate embryonic differentiation. Like PAX6,
many other PAX genes are expressed in the developing nervous
system and are believed to help regulate neurogenesis. In mice,
Pax1 is expressed in, among other areas, the developing vertebral
column and thymus; Pax3 in the early neural tube; Pax6,
upon closure of the neural tube, in the developing hindbrain
and forebrain and several other areas of the developing nervous
system; and Pax8 in the neural tube, hindbrain, and thyroid.
PAX3 mutant phenotypes include deafness, depigmentation,
and spina bifida (failure of neural tube closure) (Strachan, et
Nine unlinked PAX genes have been identified by their homology
to the paired (prd) segmentation gene expressed in
Drosophila larvae. These genes encode proteins that all include
a 128-amino acid sequence-specific DNA-binding domain, the Paired
box, which can regulate the expression of other genes. Recent
research on the binding of the Pax6 paired domain has revealed
a possible structure of three a-helixes (Strachan, et al. 1994),
a consensus DNA-binding sequence, and evidence for conformation
changes in the protein upon binding (Epstein, et al. 1994).
PAX genes (4,6,3,7), also contain another common
DNA-binding element, the homeobox. The homeobox, first discovered
in Drosophila, encodes a 60-amino acid homeodomain that is
thought to be part of more than 0.2% of the total number of vertebrate
genes. The homeodomain, too, contains three a-helices, one of which
is responsible for target sequence recognition (Glaser, et al. 1992).
Recent research has also shown that the paired domain and homeodomain
may interact cooperatively to recognize multiple DNA binding sites.
(Jun and Desplan, 1996). Unlike the HOX (Homeo
boX) family of homeobox-containing genes, which regulate
many aspects of embryonic morphogenesis, PAX genes are not
clustered but are dispersed throughout the genome (Mark, et al.
1997). Particular attention has been devoted to the molecular biology
of the PAX6 gene because of its high degree of conservation
and its seeming ability to regulate development of both the compound
and the simple eye structures.
PAX6 Gene Structure
Human PAX6 is transcribed as a 2.7kb mRNA and encodes a
422-amino-acid protein that includes the paired box, the homeo box,
and a third possible DNA-binding motif, the PST domain (Proline,
Serine, and Threonine-rich sequence; Glaser, et al. 1995. See Figure
1). Interestingly, PAX6 contains an alternative mRNA splice-site
in the paired domain which can result in a 42-nucleotide insertion;
the insertion allows the carboxy terminal subregion of the paired
domain to recognize a novel DNA sequence, allowing PAX6 to
regulate an expanded or restricted set of genes depending on how
the mRNA is spliced (Epstein, et al. 1994). PAX6 extends
over 22kb and contains 14 exons and intron sequences in the homeobox
itself. In addition, a CCAGCATGC translation start site in exon
4, a TAA stop codon in exon 13, a transcription start site and promoter
region with TATA, CAAT, and GC regulatory elements, and three possible
polyadenylation signals have all been characterized in several converging
lines of research. (Glaser, et al. 1992).
1. Characterization of human PAX6 cDNA. A, Hatched and
solid areas represent the paired and homeo domains, respectively.
The C-terminal segment is rich in proline, serine, and threonine
residues (PST domain). Individual exons are numbered and the position
of each exon boundary is marked by a vertical line. The AUG initiation
codon, TAA stop codon, and poly-A (24 adenosine residues) are indicated.
B, Comparison of the human PAX6 and zebrafish pax(zf-a) protein
sequences. Predicted ?-helices in the paired and homeodomain are
overlined and the positions of splice junctions are indicated by
triangles. The alternative peptide encoded by exon 5a is inserted
at the asterisk. The overall amino acid homology between PAX6 and
pax (zf-a) is 96.0 percent. Figure used with author's and original
journal's permission (Glaser, et al., 1992).
Mutations in various positions within PAX6 give rise to gene
dosage effects that support the hypothesis that PAX6 regulates
gene expression during development by means of concentration gradients
with other transcription factors. In one family, truncation of PAX6
in the PST domain by a point mutation in exon 12 led to cataracts
and decreased visual acuity in the father; truncation of PAX6
in the paired domain by a point mutation in exon 6 led to iris absence,
cataracts, severe decreased visual acuity, and other ocular malformations
in the mother; and a daughter compound heterozygote with a copy
of each of the parent's mutated PAX6 genes died eight days
after birth with severe central nervous system and craniofacial
defects and anopthalmia (Glaser, et al. 1994).
Drosophila as a PAX6 model system
Research on PAX6 has been facilitated by the discovery of
a PAX6 homologue in Drosophila, the eyeless (ey)
gene (Quiring, et al. 1994). Mutations in ey produce eye
defects similar to those produced by Sey (mouse) and PAX6
mutations. Drosophila provides a convenient and useful
model for PAX6 because its growth is rapid, its genetic and
embryonic mechanisms have been well-characterized, and it is simple
and inexpensive to maintain. Like PAX6, ey is expressed
in the embryonic nerve cord, specific regions of the brain, and
in eye precursors.
Much PAX6 research focuses on the developmental pathways
that lead to eye formation. Upstream and downstream regulatory
genes have been investigated for ey in Drosophila.
Hypothesized downstream targets of ey include eyes absent,
sine oculis, and dachshund (Halder, et al. 1995).
Though few upstream regulatory protein products of PAX6 or
ey have been identified, a number of regulatory elements in the
PAX6 and ey genes have been identified through gel-shift
and footprinting assays and in transgeneic in vivo studies
of mice expressing a lacZ reporter under the control of various
Pax6 regulatory elements (R. Maas, personal communication).
Other possible upstream regulators of Pax6 include activin
A (Pituello, et al., 1995) and sonic hedgehog (another developmental
control gene) (Ericson, et al., 1997).
PAX6 as a master regulator
Because ey expression is not affected by mutations in other
eye-determining genes, and because PAX6 is so highly conserved,
PAX6 is hypothesized to be a "master regulator of eye development"
(qtd. in Displan, 1997). This hypothesis was recently supported
by the finding in Drosophila that misexpression of ey
could induce ectopic eye formation on appendages (Halder, et al.
1995). The ectopic eyes were fully formed and included the full
complement of cell types and structures, including photoreceptors.
Later research, however, revealed that genes supposedly "downstream"
of ey could also induce ectopic eyes. The products of Sine
oculis (so), which encodes a homeobox-like domain, and eyes
absent (eya), which encodes a novel nuclear protein, form a
complex and can induce ectopic yes similar to those formed
by ectopic ey expression (Pignoni, et al. 1997). More significantly,
so and eya could together induce ey expression,
which is not consistent with the idea of ey as the master
regulatory of those genes. In addition, dachshund (dac),
which encodes another novel nuclear protein and is induced by ey
expression, and eya misexpression resulted in full ectopic
eye production as well, while dac and eya alone could
each weakly induce ectopic eye formation (Chen, et al. 1997).
These gain-of-function experiments suggest that the protein products
of ey and of its human homologue PAX6 operate not
in a hierarchical linear pathway, but as a network with numerous
feedback loops. A second possible hypothesis is that, because eye
regulatory genes are activated several times during development,
they are turned on in sequence at each stage. The repeated use of
the same regulatory genes during eye development has been explained
in conjunction with the high degree of conservation of the genes:
as eye formation progressed during evolution from simple photoreceptors
to the complex visual systems in insects and vertebrates, the same
regulatory genes were co-opted for each new developmental pathway
It is also possible that PAX6 and other eye development
genes play a role in the development of other organs, such as the
pancreas (St-Onge, et al., 1997). In addition, an eya homologue
in humans, when mutated, has no apparent affect on eye formation.
These results suggest that PAX6 may be involved in the larger
process of organogenesis rather than only oculogenesis.
The highly conserved PAX6 and its Drosophila homologue
ey are key players in a highly complex developmental pathway
leading to formation of both simple and compound eyes and possibly
other organs as well. Research in several laboratories is directed
towards characterizing the complex network of regulatory genes involved.
Several PAX6 enhancer elements show promise as sites for
upstream regulation of PAX6, and possibly even downstream
products of eya, so, or dac may play a regulatory
Several medical applications have arisen in recent years based
on research on the PAX6 gene family. Because the gene
has been sequenced, prenatal diagnosis of aniridia is now possible.
In addition, some evidence suggests that PAX6 may be expressed
by damaged eye tissue to induce limited regeneration; artificial
upstream regulation of PAX6 may eventually be used to induce
such regeneration. Finally, some cancers, including alveolar rhabdomyosarcoma,
may be caused by PAX mutations (Strachan, et al. 1994). These
findings suggest potential therapeutic applications for PAX6
research and may lead to a more complete understanding of its role
in eye development.
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Journal of Young
Investigators. 1998. Volume One.
Copyright © 1998 by Adam A. L. Friedman and JYI. All rights