Role of Pax genes in eye evolution: a cnidarian PaxB gene uniting Pax2 and Pax6 functions

PaxB from Tripedalia cystophora, a cubomedusan jellyfish possessing complex eyes (ocelli), was characterized. PaxB, the only Pax gene found in this cnidarian, is expressed in the larva, retina, lens, and statocyst. PaxB contains a Pax2/5/8-type paired domain and octapeptide, but a Pax6 prd-type homeodomain. Pax2/5/8-like properties of PaxB include a DNA binding specificity of the paired domain, activation and inhibitory domains, and the ability to rescue spa(pol), a Drosophila Pax2 eye mutant. Like Pax6, PaxB activates jellyfish crystallin and Drosophila rhodopsin rh6 promoters and induces small ectopic eyes in Drosophila. Pax6 has been considered a "master" control gene for eye development. Our data suggest that the ancestor of jellyfish PaxB, a PaxB-like protein, was the primordial Pax protein in eye evolution and that Pax6-like genes evolved in triploblasts after separation from Cnidaria, raising the possibility that cnidarian and sophisticated triploblastic eyes arose independently.     

Duplicated Pax6 genes in Glomeris marginata (Myriapoda: Diplopoda), an arthropod with simple lateral eyes

Composite (facetted) eyes comprised by several units, termed ommatidia, are an ancestral feature in the arthropods. Some arthropods, however, do not possess composite eyes, obviously by secondary reduction. Reductions on the level of conserved eye developmental genes are one possibility to reduce the visual system. The genes of the Pax6 family have been shown to be key regulators of visual system development in a wide variety of animals. Reduction of Pax6 expression may therefore be expected in a species with reduced eyes. Here I have investigated the myriapod Glomeris marginata that displays very simple eyes. Glomeris, however, possesses two Pax6 genes that, based on their sequence, are similar to Drosophila eyeless (ey) and twin of eyeless (toy), respectively. Both genes are highly expressed in the optic lobes and the ventral nerve cord of developing embryos. Furthermore, homologs of other high-ranking eye developmental genes like hedgehog, decapentaplegic, dachshund, and homothorax are expressed in the optic lobes. This indicates that eye reduction in Glomeris is not realized at the level of the Pax6 genes or other genes on the upper levels of the eye development network. I suggest instead that the simple eyes of Glomeris are the product of changes at a much lower level in the network, probably at the level of genes directly regulating ommatidia development or ommatidia number and arrangement.     

Multifocal lenses compensate for chromatic defocus in vertebrate eyes

The focal length of the vertebrate eye is a function of wavelength, i.e. the eye suffers from longitudinal chromatic aberration. Chromatic defocus is a particularly severe problem in eyes with high light-gathering ability, since depth of field is small due to a pupillary opening that is large in relation to the focal length of the eye. Calculations show that in such eyes only a narrow spectral band of light can be in focus on the retina. For the major part of the visual spectrum, spatial resolution should be limited by the optics of the eye and far lower than the resolving power achievable by the retinal cone photoreceptor mosaic. To solve this problem, fishes with irises unresponsive to light have developed lenses with multiple focal lengths. Well-focused images are created at the wavelengths of maximum absorbance of all spectral cone types. Multifocal lenses also appear to be present in some terrestrial species. In eyes with mobile irises, multifocal lenses are correlated with pupil shapes that allow all zones of the lens, with different refractive powers, to participate in the imaging process, irrespective of the state of pupil constriction. Accepted: 6 November 1998     

Pax6 heterozygous eyes show defects in chamber angle differentiation that are associated with a wide spectrum of other anterior eye segment abnormalities

The development of the chamber angle was studied in the eyes of heterozygous Pax6(lacZ/+) mutant mice (Nature 387 (1997) 406). Mutations in PAX6 cause aniridia, a condition that is frequently associated with glaucoma, a blinding disease that may be associated with chamber angle defects. Mesenchymal cells were seen in the chamber angle at P1-P5. In wild-type mice, these cells differentiated into typical trabecular meshwork (TM) cells next to Schlemm's canal. In Pax6(lacZ/+) mice, TM cells remained undifferentiated and Schlemm's canal was absent. From P1 to P4, staining for beta-galactosidase and immunoreactivity for Pax6 were observed in chamber angle mesenchyme, but were absent later. Cultured murine TM cells expressed Pax6. The defects in chamber angle and TM differentiation were associated with a wide spectrum of other anterior eye defects, which included various degrees of iris hypoplasia and corneal haze, isolated iridocorneal adhesions and atypical coloboma, and a vascularized cornea in all adult animals. A third of the animals showed Peters' anomaly including corneal opacity and iridocorneal adhesions. The separation of the lens from the cornea was incomplete, and epithelial layers of lens and cornea were continuous. Pax6 activity is directly required for differentiation of the chamber angle. Variations in phenotype of Pax6(lacZ/+) mice appear not to involve direct dominant-negative or dose-dependent effects.     

Pax6 and eye development in Arthropoda

The arthropod compound eye is one of the three main types of eyes observed in the animal kingdom. Comparison of the eyes seen in Insecta, Crustacea, Myriapoda and Chelicerata reveals considerable variation in terms of overall cell number, cell positioning, and photoreceptor rhabdomeres, yet, molecular data suggest there may be unexpected similarities. We review here the role of Pax6 in eye development and evolution and the relationship of Pax6 with other retinal determination genes and signaling pathways. We then discuss how the study of changes in Pax6 primary structure, in the gene networks controlled by Pax6 and in the relationship of Pax6 with signaling pathways may contribute to our insight into the relative role of conserved molecular-genetic mechanisms and emergence of evolutionary novelty in shaping the ommatidial eyes seen in the Arthropoda.     

Role of Pax6 in development of the cerebellar system.

Post-mitotic neurons generated at the rhombic lip undertake long distance migration to widely dispersed destinations, giving rise to cerebellar granule cells and the precerebellar nuclei. Here we show that Pax6, a key regulator in CNS and eye development, is strongly expressed in rhombic lip and in cells migrating away from it. Development of some structures derived from these cells is severely affected in Pax6-null Small eye (Pax6(Sey)/Pax6(Sey)) embryos. Cell proliferation and initial differentiation seem unaffected, but cell migration and neurite extension are disrupted in mutant embryos. Three of the five precerebellar nuclei fail to form correctly. In the cerebellum the pre-migratory granule cell sub-layer and fissures are absent. Some granule cells are found in ectopic positions in the inferior colliculus which may result from the complete absence of Unc5h3 expression in Pax6(Sey)/Pax6(Sey) granule cells. Our results suggest that Pax6 plays a strong role during hindbrain migration processes and at least part of its activity is mediated through regulation of the netrin receptor Unc5h3.     

Pax 6: mastering eye morphogenesis and eye evolution.

Pax 6 genes from various animal phyla are capable of inducing ectopic eye development, indicating that Pax 6 is a master control gene for eye morphogenesis. It is proposed that the various eye-types found in metazoa are derived from a common prototype, monophyletically, by a mechanism called intercalary evolution.     

Intestinal tumorigenicity of mice carrying hemizygous Pax6, Pax6(Sey-4H).

The genotype-phenotype relationship in mice was examined experimentally using one of the small eye mutants, Pax6(Sey-4H), which deletes the chromosome 2 middle region, hemizygously. The genotyping indicated that the deleted region starts at a site 102.60 Mb from the centromere and has a length of 6.51 Mb, in which 12 known and 27 novel genes are located. Expecting the development of myeloid leukemia, gamma-irradiation was performed to female mutants at the age of 10 weeks. The mutants did not develop myeloid leukemia during the observation period of 18 months. Instead, they developed tumors in the alimentary tract spontaneously (56.0%). The tumor latency was shortened by the radiation exposure, but the tumor incidence of the gamma-irradiated group (62.5%) was as high as that of spontaneously developing tumors. Intraductal proliferation of the epithelium of the Wirsung duct was observed in the gamma-irradiated mutants (18.8%). Considering the results of the Pax6(Sey-4H) mutant together with those of another small eye mutant, Pax6(Sey-3H), the anomaly and the tumorigenicity of the intestinal tract were closely related to the hemizygosity of the 3.2 Mb segment of chromosome 2, where both mutants show a common deletion.     

Pax 4 and 6 regulate gastrointestinal endocrine cell development.

The mechanisms behind the cell-specific and compartmentalized expression of gut and pancreatic hormones is largely unknown. We hereby report that deletion of the Pax 4 gene virtually eliminates duodenal and jejunal hormone-secreting cells, as well as serotonin and somatostatin cells of the distal stomach, while deletion of the Pax 6 gene eliminates duodenal GIP cells as well as gastrin and somatostatin cells of the distal stomach. Thus, together, these two genes regulate the differentiation of all proximal gastrointestinal endocrine cells and reflect common pathways for pancreatic and gastrointestinal endocrine cell differentiation.     

The Optimedin gene is a downstream target of Pax6

The Optimedin gene, also known as Olfactomedin 3, encodes an olfactomedin domain-containing protein. There are two major splice variants of the Optimedin mRNA, Optimedin A and Optimedin B, transcribed from different promoters. The expression pattern of the Optimedin A variant in the eye and brain overlaps with that for Pax6, which encodes a protein containing the paired and homeobox DNA-binding domains. The Pax6 gene plays a critical role for the development of eyes, central nervous system, and endocrine glands. The proximal promoter of the Optimedin A variant contains a putative Pax6 binding site in position -86/-70. Pax6 binds this site through the paired domain in vitro as judged by electrophoretic mobility shift assay. Mutations in this site eliminate Pax6 binding as well as stimulation of the Optimedin promoter activity by Pax6 in transfection experiments. Pax6 occupies the binding site in the proximal promoter in vivo as demonstrated by the chromatin immunoprecipitation assay. Altogether these results identify the Optimedin gene as a downstream target regulated by Pax6. Although the function of optimedin is still not clear, it is suggested to be involved in cell-cell adhesion and cell attachment to the extracellular matrix. Pax6 regulation of Optimedin in the eye and brain may directly affect multiple developmental processes, including cell migration and axon growth.