Drosophila is a genus of flies that is commonly used as a model organism to study a variety of biological processes. The eyeless gene in Drosophila encodes a transcription factor. It is homologous (has a similar structure) to the Pax-6 gene and Aniridia gene in mice and humans respectively. Aniridia, Pax-6, and eyeless are all expressed during eye development. Loss-of-function mutations in these genes result in eye structures becoming smaller or being lost all together, suggesting that these genes are important in eye development. In this study, the researchers were able to target eyeless expression in imaginal disc primordia (a part of insect larva that gives rise to the outside of an adult insect). This resulted in the appearance of eye structures in places where eyes wouldn’t normally appear, including wings, legs, and antennae. These eyes were normal and even had fully developed ommatidia (units that make up compound eyes in insects) and photoreceptor cells, which aid in vision. These results show that eyeless is the master control gene for eye development.
The researchers wanted to see if they induced the expression of eyeless where it is not usually expressed, what would happen. They also wanted to understand whether eyeless was the master control gene for eye development.
Mutations in eyeless result in partial or complete absence of eyes. Sequencing of eyeless has shown that it encodes a transcription factor. Eyeless is homologous to Pax-6 in mice and Aniridia in humans, sharing over 90%% of their sequence identities. In addition, several splice sites are conserved, indicating that these genes evolved from a common ancestor.
Both Pax-6 and eyeless have similar expression patterns during development. In mice, Pax-6 expression is observed in the spinal cord, some regions of the brain, and the eyes. It is expressed in the eyes from the beginning to the end of eye development. In Drosophila, eyeless is expressed in the ventral nerve cord (analogous to the spinal cord) and in the brain. Later, it is expressed in structures that give rise to the eyes. It continues to be expressed throughout eye development.
Because mutations in eyeless and Pax-6 result in the reduction or complete absence of eye structures, it has been suggested that they are the master control genes in eye development. Mutations in other genes associated with eye development do not affect the expression of eyeless, indicating that eyeless is expressed upstream of these other genes.
Master control genes can be detected by their mutant phenotype (the set of observable characteristics resulting from genetic expression). Mutations in homeotic genes (genes that give rise to particular body segments or structures) have identified master control genes. These genes are characterized by a DNA sequence called a homeobox. Loss- and gain-of-function mutations result in opposite phenotypes. In addition, ectopic expression of these genes result in ectopic phenotypes. For example, ectopic expression of a gene called Antp has induced the formation of legs where antennae should be.
Eyeless is different from homeotic genes in that loss-of-function mutations result in the loss of eye structures rather than a transformation. This shows that eyeless is required for eye development. If eyeless is the master control gene for eye development, ectopic expression of eyeless should induce the formation of eye structures on other parts of the body. To test this hypothesis, the researchers used the GAL4 system, a biochemical method used to study gene expression and function, to ectopically express eyeless.
Induction of ectopic eye structures
Ectopic eye structures were induced in wings, legs, antennae, and halteres in Drosophila. The eye structures bulged out of the tissue in which they were induced, which could have resulted from minimizing contact surface between the eye tissue and non-eye tissue types. In some cases, ectopic eye development interfered with the differentiation of surrounding cells, resulting in less-developed eye structures. Well-developed eyes were most frequently observed on antennae and wings, but in some cases there were defects. Eye structures on the legs were smaller, but were relatively normal.
Photoreceptors in the ectopic eyes
Analysis of the ectopic eye structures showed identical cells and structures in normal eyes. The researchers were able to distinguish various structures in the eye required for vision, including photoreceptor cells. The researchers analyzed nerve cell differentiation in these photoreceptors. The sequence of differentiation was the same in the ectopic eyes and normal eyes. This suggests the development of these ectopic eyes proceeds normally. Taken together, these observations show that eyeless can induce the formation of complete and developmentally normal eyes. It is unknown whether these eyes are functional.
Role of eyeless in eye morphogenesis
The findings above indicate that eyeless is the master control gene for eye development, since it can induce ectopic eye structures. Eyeless is expressed first and controls a set of subordinate genes in eye development in a regulatory cascade. Eyeless may control later steps in eye development as well, being reused in different steps of eye development. This is supported by the fact that eyeless controls other functions besides eye development in the nervous system, and loss-of-function mutations in eyeless are lethal. The loss of eye structures alone is not the cause of lethality, so the lack of development in other structures controlled by eyeless must contribute to this lethality.
The transformation of antennal, leg, and wing tissue into eye structures by eyeless expression suggests that eyeless is a homeotic gene.
The high degree of conservation between Pax-6, Aniridia, and eyeless in their genetic sequences, phenotypes, and expression patterns suggest that eyeless is a master control gene for eye development in invertebrates and vertebrates. This suggests that genetic control mechanisms of development are much more universal than previously thought. The researchers call for more research comparing the regulatory cascade in Drosophila eye development with that in mice to find out what the differences are and how new genes have been incorporated over the course of evolution.
The GAL4 system was used to ectopically express eyeless. GAL4 can activate transcription of any gene if it is preceded by GAL4 binding sites.