Molecular Regulation of Eye Development: Role of the PAX Gene
Corresponding author: Dr. Tabinda Hasan, Anatomy department, Al Maarefa college of medicine, Riyadh, KSA,
The eye develops from an out-pouching of the diencephalon. A series of induction and regionalization events in the neuro-ectoderm generate and shape the optic cup, vesicle, lens etc. The origin of Pax(paired box protein) genes predates the origin ofeyes and even the nervous system. The ancestral Pax gene (PaxB) was related to Pax2,5 and 8. Interestingly, it was identified in the sponge that has neither eyes nor the nervous system. Cnidaria are the most basal animal models that have simple or complex (lens containing) eyes as well as Pax genes. Cnidarian (Tripedalia) PaxB gene is expressed in lens and retina and activates both lens crystallin and opsin reporter genes. This data indicates that modern the Pax2 and Pax6 genes evolved from a cnidarian PaxB ancestor by the mechanism of duplication and diversification in Bilateria with Pax2 loosing its HD and PAX6loosing its octapeptide (yellow box) along the way. Aniridia, a human genetic disease manifested by alterations in structure and function of the eye, includes reduced iris size, absence of fovea, and lens deformities.  It was documented as a genetic disease more than a hundred years ago. It is an ideal model for demonstrating autosomal dominant genetic disorders because of the high penetrance of its mutant alleles, the ease of diagnosis at birth, and a relatively similar incidence in various populations.  Recently, the aniridia gene (AN) was mapped (to chromosome band 11p13). Pax-6, also called the aniridia type II protein (AN2) or oculorhombin is a protein that is encoded by the PAX6 gene in humans. The gene PAX6 was determined to be the key regulator of eye development as well as that of the central nervous system, with researches proving that PAX2 expresses more in the optic stalk and the PAX6 expressing predominantly in the optic cup . Many studies that led to the identification of PAX6 as the human aniridia gene were conducted in lower mammals. These studies demonstrated PAX6as a very sophisticated gene, that is highly conserved among vertebrates and lower animals. The PAX6 ‘dosage effect’ in cases of aniridia ranges from mild loss of visual acuity and cataracts to severe nervous system defects and synopthalmia (conjointeyes) or anopthalmia (complete absence of the eyes)  PAX6 homologues have been located in Drosophila fly (eyeless), with similar functions and similar mutant phenotypes. The amino acid sequence identities of approximately 90% are found .PAX6 is 96% identical in its amino acid sequence to pax(zf-a) in zebrafish; but the two species diverged over 400 million years . This degree of conservation parallels that of histones, one of the most highly conserved proteins known. The knowledge of the mechanism of PAX6 explained unique evolution trends that Darwin had not been able to. In his notable work, ’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 these structures might have developed separately through the mechanism of ‘convergent evolution’ . However, in both insects and vertebrates, PAX6 is expressed in the embryo just prior to the formation of the optic vesicle. It has been found that mis-expression of a PAX6 homologue in flies and chicks can induce ectopic eyes. Based on this evidence, it
has been speculated that PAX6 is a master regulatory gene that induces eye development in a broad range of animals .
The multi-gene family of PAX
PAX6 belongs to the PAX multigene family of transcription factors that help regulate embryonic differentiation. These genes encode proteins that include a 128-amino acid sequence-specific DNA-binding domain, the Paired box, which can regulate the expression of other genes. Recent research on Pax6 paired domain binding has revealed a possible structure of three A-helixes , a consensus DNA-binding sequence, and evidence for conformation changes in protein binding . Nine unlinked PAX genes have been identified in Drosophila larvae. The PAX6 protein is one among the many transcription factors that induce embryonic differentiation along the major body axis. These transcription factors bind to specific DNA sequences of other genes in response to concentration gradients of regulatory proteins, and adjust their expression; thus translating positional information into developmental patterns for distinct structures . Like PAX6, many other PAX genes are expressed in the developing nervous system. These are believed to regulate neurogenesis. In mice, Pax1 is expressed in the developing vertebral column and thymus; Pax3 in the early neural tube; Pax6, in the developing hindbrain and forebrain upon closure of the neural tube, Pax8 in the neural tube, hindbrain, and thyroid. PAX3 mutants result in deafness, depigmentation, and spina bifida (failure of neural tube closure) . PAX genes number 3,4,6,7 also contain another common DNA-binding element, the homeobox(HOX). The homeobox was first discovered in Drosophila. It encodes a 60-amino acid homeo-domain that is known to be part of more than 0.2% of the total vertebrate genes. The homeo-domain also contains three A-helices, one of which is responsible for target sequence recognition . Recent research has shown that the Pax 6 paired domain and homeo-domain may interact cooperatively to recognize multiple DNA binding sites . Unlike the HoX family of homeobox-containing genes, which regulate many aspects of embryonic morphogenesis, PAX genes are not clustered but are dispersed randomly throughout the genome .
Structure of the PAX6 Gene
Human PAX6 is transcribed as 2.7kb mRNA and encodes a 422-amino-acid protein. This protein includes the paired box, the homeo box, and a DNA-binding motif called the PST domain-Proline, Serine, and Threonine-rich sequence . (Figure1). Interestingly, PAX6 contains an alternative mRNA splice-site in the paired domain which can result in a 42-nucleotide insertion. This insertion allows the carboxy terminal subregion of the paired domain to recognize a novel DNA sequence, therefore allowing PAX6 to regulate an expanded or restricted set of genes depending upon how the mRNA is spliced (Epstein, et al. 1994). PAX6 extends over 22kb and contains 14 exons and intron sequences in the homeobox. Additionally, there is 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 atleast three possible polyadenylation signals in several converging lines of research. Mutations at various locations within the PAX6 gene give rise to “dosage effects” that support the hypothesis thatPAX6 regulates gene expression during eye development through concentration gradients with other transcription factors. In a popular study by Glaser on a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects, it was found that 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, severely decreased visual acuity, and other ocular malformations in the mother; and a compound heterozygote daughter with a copy of each of the parent’s mutated PAX6 genes died eight days after birth with severe central nervous system and craniofacial anomalies. Research on PAX6 was facilitated by the discovery of a PAX6 homologue in Drosophila,called the eyeless (ey) gene . Mutations in ey produced eye defects similar to those produced by Sey in mouse and PAX6 mutations in humans. Drosophila provides a convenient 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 nerve cord, specific regions of the brain, and in embryonic eye precursors. Most researches on the PAX6 focus on developmental pathways that lead to eye formation. Upstream and downstream regulatory genes have been investigated for ey in Drosophila. A number of regulatory elements in the PAX6 and ey genes have been identified through gel-shift and foot-printing assays and transgeneic in vivo studies in mice expressing a lacZ reporter under the control of various PAX6 regulatory elements. The popular hypothesized downstream targets of ey include eyes absent, sine oculis, and dachshund . Relatively few upstream regulatory protein products of PAX6 or ey have been identified. Possible upstream regulators of PAX6 include activin A  and sonic hedgehog (SHH), another developmental control gene that produces separation of the eye primordial into two separateeye fields . Defects in SHH signalling lead to conditions like synopthalmia.
The vertebrate PAX6 locus encodes three different protein isoforms-the canonical PAX6, the PAX6 (5a), and the PAX6 (ΔPD).The canonical PAX6 protein includes an N-terminal paired domain, connected by linker regions to paired-type homeodomain,and to a proline/ serine/ threonine (P/S/T)-rich C-terminal domain. Each of the paired domain and pairedtype homeodomain have DNA binding activities, whereas the P/S/T-rich domain possesses a transactivation function . PAX6 (5a), being a product of the alternatively spliced exon 5a, results in a 14 residue insertion in the paired domain that alters the specificity of this DNA binding activity. The nucleotide sequence corresponding to the linker region encodes a set of three alternative translation start codons from which the third PAX6 isoform originates. Collectively, these are known as the PAX6 (ΔPD) or pairedless isoforms. These three gene products lack a paired domain. The paired less proteins have molecular weights of 43, 33, or 32kDa, depending upon the particular start codon used. PAX6 transactivation function is attributed to the variable length C-terminal P/S/T-rich domain that stretches 153 residues long in human and mouse proteins.
‘Gain of function mechanisms’ showing PAX6 as a master regulator
PAX6 is hypothesized to be a “master regulator of eye development”, because ey expression is not affected by mutations inother eye-determining genes, and also because PAX6 is so highly conserved . A “knockout” model was created using miceduring the point of time when the mouse did not express PAX6. This ‘knockout” model is eyeless or has very underdevelopedeyes further indicating PAX6 is required for proper eye development. This hypothesis was recently supported by the findingthat misexpression of ey could induce ectopic eye formation on appendages in Drosophila . Interestingly, these ectopic eyes were fully formed and included the full complement of cell types and structures, including photoreceptors. Later research revealed that genes falling “downstream” of ey could also induce ectopic eyes. The products of Sine oculis (so), and eyes absent (eya), form a complex that can induce ectopic eyes similar to those formed by ectopic ey expression . Moresignificantly, so and eya could together induce ey expression, which is not consistent with the idea of ey being the masterregulator of those genes. Additionally, dachshund (dac); which is induced by ey expression and encodes a novel nuclearprotein, and eya mis-expression resulted in full ectopic eye production as well, whereas dac and eya alone could each weaklyinduce ectopic eye formation . These experiments suggest that the protein products of ey and its human homologue PAX6operate not in a hierarchical dogmatic pathway. Rather, they operate as a network with numerous feedback loops. Anotherhypothesis is that, because eye regulatory genes are activated several times during development, they are turned on and off 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. During evolution, as eye formation progressed from simple photoreceptors to complex visual systems in insects and vertebrates, the same regulatory genes were co-opted for each new developmental pathway for the logic of convenience in familiarity . It could well be likely, that PAX6 and other eyedevelopment genes also play a role in the development of other unrelated organs, such as pancreas and olfactory system .PAX6 may be involved in the larger process of organogenesis rather than only oculogenesis. The highly conserved PAX6 and itsDrosophila homologue ey are key regulators in the highly complex developmental pathway leading to the formation of both simple and compound eyes and possibly other organs as well in lower animals and humans. Currant researches are directed towards characterizing the complex network of regulatory genes involved. Several PAX6 enhancer elements for upstream regulation of PAX6, and possibly even downstream products of eya, so, or dac may play a regulatory role. In addition, there is limited evidence suggesting that PAX6 may be expressed by the damaged eye tissue to induce limited regeneration. Artificial upstream regulation of PAX6 may eventually induce such regeneration. Finally, certain cancers, including alveolar rhabdo-myosarcoma, may be caused by PAX mutations.
Pax genes are developmental control genes that have a prominent role in encoding transcription factors. Their fundamentalrole in animal development has been well illustrated by genetic studies. Loss-of-function mutations cause specific developmental defects. Additionally, gain-of-function mutations have been implicated in cancer. For a fuller insight into the molecular mechanisms underlying Pax gene function, it is necessary to define Pax target genes, recognize proteins that modulate properties of Pax transcription factors by direct physical interaction and expose the basis of the haploinsufficiency phenomenon. With their multiple functions during development in various animal phyla, Pax genes can serve as a paradigm ofhow transcription factors control development. In addition, due to high Pax gene conservation throughout the metazoankingdom it is possible to understand how the individual gene functions have been modified during the course of evolution. In fact, by focusing on one gene family in well-defined systems one can hope to contribute towards a better understanding of the sophisticated mechanisms underlying evolution, development and disease in general. In recent years, several medicalapplications have arisen based on researches on the PAX6 gene family. Because of gene sequencing, prenatal diagnosis of aniridia is now possible. Further research is needed to explore the full potential of therapeutic applications of PAX6 and a more comprehensive understanding of its role in eye development.
3. Numayama-Tsuruta K, Arai Y, Takahashi M, Sasaki-Hoshino M, Funatsu N et al. Downstream genes of Pax6 revealed by comprehensive transcriptome profiling in the developing rat hindbrain. BMC Dev Biol. 2010, 10:6.
7. Machon O, Kreslova J, Ruzickova J, Vacik T, Klimova L et al. Lens morphogenesis is dependent on Pax6-mediated inhibition of the canonical Wnt/beta-catenin signaling in the lens surface ectoderm. Genesis. 2010, 48(2): 86-95.
10. Favor J, Peters H, Hermann T, Schmahl W, Chatterjee B et al. Molecular characterization of Pax6(2Neu) through Pax6(10Neu): an extension of the Pax6 allelic series and the identification of two possible hypomorph alleles in the mouse Mus musculus. Genetics. 2001, 159(4): 1689-1700.
11. Walcher T, Xie Q, Sun J, Irmler M, Beckers J et al. Functional dissection of the paired domain of Pax6 reveals molecular mechanisms of coordinating neurogenesis and proliferation. Development. 2013, 140(5): 1123-1136.
12. Ninkovic J, Steiner-Mezzadri A, Jawerka M, Akinci U, Masserdotti G et al. The BAF Complex Interacts with Pax6 in Adult Neural Progenitors to Establish a Neurogenic Cross-Regulatory Transcriptional Network. Cell Stem Cell. 2013, 13(4):403-418.