Growth and specification: fly Pax6 homologs eyegone and eyeless have distinct functions

Development requires not only the correct specification of organs and cell types in the right places (pattern), but also the control of their size and shape (growth). Many signaling pathways control both pattern and growth and how these two are distinguished has been something of a mystery. In the fly eye, a Pax6 homolog (eyeless) controls eye specification together with several other genes. Now Dominguez et al.1 show that Notch signaling controls eye growth through a second Pax6 protein (Eyegone). In mice and humans the single Pax6 gene appears to encode both specification and growth controlling proteins through alternative mRNA splicing.     

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.     

Pax genes and eye organogenesis

Pax6 is a highly conserved gene that controls eye development in all species where it has been tested. In spite of this common 'master control regulator', the eyes of different animals are morphologically very different and it is believed that they have evolved independently multiple times through evolution. Recent works looking at eye development in 'primitive' species offer some explanation as to the surprising amount of conservation in genetic and morphogenetic pathways involved in eye development. These studies not only implicate the Pax genes but also the So/Six gene family in playing a crucial ancestral role in visual system development.     

Pax genes in development and maturation of the vertebrate visual system: implications for optic nerve regeneration

Pax genes play a pivotal role in development of the vertebrate visual system. Pax6 is the master control gene for eye development: ectopic expression of Pax6 in Xenopus laevis and Drosphila melanogaster leads to the formation of differentiated eyes on the legs or wings. Pax6 is involved in formation of ganglion cells of the retina, as well as cells of the lens, iris and cornea. In addition Pax6 may play a role in axon guidance in the visual system. Pax2 regulates differentiation of the optic disk through which retinal ganglion cell axons exit the eye. Furthermore, Pax2 plays a critical role in development of the optic chiasm and in the guidance of axons along the contralateral or ipsilateral tracts of the optic nerve to visual targets in the brain. During development Pax7 is expressed in neuronal cells of one of the major visual targets in the brain, the optic tectum/superior colliculus. Neurons expressing Pax7 migrate towards the pia and concentrate in the stratum griseum superficiale (SGFS), the target site for retinal axons. Together, expression of Pax2, 6 and 7 may guide axons during formation of functional retinotectal/collicular projections. Highly regulated Pax gene expression is also observed in mature animals. Moreover, evidence suggests that Pax genes are important for regeneration of the visual system. We are currently investigating Pax gene expression in species that display a range of outcomes of optic nerve regeneration. We predict that such information will provide valuable insights for the induction of successful regeneration of the optic nerve and of other regions of the central nervous system in mammals including man.     

EGF receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification

The Drosophila compound eye is specified by the concerted action of seven nuclear factors that include Eyeless/Pax6. These factors have been called "master control" proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, we find that several of these eye specification genes are not coexpressed in the same embryonic cells-or even in the presumptive eye. We demonstrate that the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes, and show that specification occurs much later than previously thought-not during embryonic development but in the second larval stage.     

The genetic control of eye development and its implications for the evolution of the various eye-types

Mutations in the Pax 6 homologs of mammals and insects prevent eye development and targeted expression of both mammal and insect Pax 6 homologs is capable of inducing functional ectopic eyes. Supported by RNA interference experiments in planarians and nemerteans, these findings indicate that Pax 6 is a universal master control gene for eye morphogenesis. Since all metazoan eyes use rhodopsin as a photoreceptor molecule and the same master control gene for eye development, we postulate a monophyletic origin of the various eye types. The finding of well developed eyes in jellyfish which essentially lack a brain, leads us to propose that the eye as a sensory organ evolved before the brain which is an information processing organ. The finding of highly developed eyes with a lens, vitreous body, stacked membranes like a retina and shielding pigment in unicellular dinoflagellates, raises the possibility that the prototypic eyes might have been acquired from symbionts.     

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.     

Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes.

We have cloned a chick homologue of Drosophila dachshund (dac), termed Dach1. Dach1 is the orthologue of mouse and human Dac/Dach (hereafter referred to as Dach1). We show that chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless (ey, the Pax6 orthologue), eyes absent (eya), and dac operate together to regulate Drosophila eye development, and that ey regulates the expression of eya and dac. We find that in the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homologue). Both Pax2 and Eya1 are required for normal ear development. Our expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, we have examined the expression of mouse Dach1 in Pax6, Pax2 and Eya1 mutant backgrounds. Our results indicate that Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy.     

Distribution of Pax6 protein during eye development suggests discrete roles in proliferative and differentiated visual cells

 Although Pax6 is required during eye development in rodents and humans, little is known about the precise role of the protein in this process. To aid in the interpretation of functional studies, we have determined the precise spatial and temporal distributions of the Pax6 protein in the eye. We find that Pax6 is initially distributed contiguously throughout a large domain of the anterior neural plate of zebrafish, including the presumptive eye fields and the dorsal diencephalon. After evagination of the optic vesicle, Pax6 becomes restricted to all proliferating cells of the pigment epithelial and neural layers of the retina. Pax6 is downregulated in most cells concomitant with differentiation. However, it remains present in several mature cell types of the eye including amacrine cells and the lens and corneal epithelia. This expression is conserved across diverse vertebrate species and suggests that Pax6 has additional conserved functions in the mature eye. Received: 27 August 1996 / Accepted: 21 October 1996