The eyes have it: genetic, morphological and functional analysis of differences in compound eyes between Drosophila species

Vision is one of the major sensory systems that helps animals navigate through the environment. There is great variation among the eyes of animals reflecting evolutionary adaptations in behaviour and habitat. Two major parameters that determine the quality of vision are: 1) How easily can objects be discriminated at different levels of light luminescence (contrast sensitivity) and 2) how far apart do two objects have to be to be detected discriminated (acuity). Contrast sensitivity depends on the amount of light absorbed by the eye's photoreceptors and therefore on the size of the eye and photoreceptor sensitivity. Acuity on the other hand depends on the density of those light-sensitive photoreceptors, for example, the density of rods and cones in the retina of the human eye. The same rules generally apply to the compound eyes of insects, which are made up of smaller eyes, called ommatidia. Larger ommatidia with larger lens diameters to collect photons gives higher contrast sensitivity. However, acuity depends on the density of ommatidia - more ommatidia per eye area gives higher acuity, and therefore there can be a trade off between contrast sensitivity and acuity determined by the size and number of ommatidia. The size of ommatidia and overall eye size varies greatly among insects, but we still know very little about the genes underlying these evolutionary differences, how this changes the development of eyes, and how these changes in eye morphology alter the vision of these animals. We have found that the gene orthodenticle (otd) causes differences in ommatidia diameter and overall eye size between the closely related species Drosophila mauritiana and Drosophila simulans. We now propose to use a range of approaches to understand how otd regulates ommatidia size and how this gene makes larger eyes in D. mauritiana, and the consequences for their vision. We will first further study the role of otd in specification of ommatidia size in the model organism D. melanogaster to learn more about the mechanisms involved. We will then put the D. mauritiana version of otd into a D. simulans genetic background and vice versa, resulting in a D. simulans fly with a D. mauritiana-like eye and the other way around. These flies can then be compared to pure D. simulans and D. mauritiana flies in their regulation of eye development, eye morphology, neuronal activity and their actual vision. The results of these experiments will reveal how natural genetic variation alters eye development to produce eyes with different optics and how this affects the vision of these flies. This will help us to better understand insect vision generally and provide new insights into how the great diversity of insect eyes and the vision of these animals have evolved. In addition, since there are major similarities between invertebrate and vertebrate eye development, including important roles of otd and its homologues, this project could provide new information about eye development more broadly in animals including humans.

Insect eyes are composed of light sensing units called ommatidia, which vary greatly in size within eyes and among species. This has important implications for vision: large ommatidia collect more photons providing greater contrast sensitivity, but this can increase interommatidial angles and lower acuity. Little is known about how ommatidia size is specified nor how genetic and developmental changes lead to differences in ommatidia size and how these changes affect vision. We found that Drosophila mauritiana has larger eyes than its sibling species D. simulans caused by wider ommatidia in the anterior region of the eye. We mapped this difference to orthodenticle (otd) and found that this gene is more highly expressed in D. mauritiana. This presents an excellent opportunity to characterise the underlying genetic and developmental basis for natural variation in ommatidia size and study consequences for vision. We will first investigate the role of otd in ommatidia size specification in D. melanogaster to better understand the underlying mechanisms. We will then introgress tagged D. simulans otd into D. mauritiana and vice versa. This will allow us to characterize the expression of otd during ommatidial maturation, the effect of both otd alleles on ommatidial development and how this is achieved through changed expression of downstream target genes using RNA-Seq. We will then utilise D. melanogaster to further investigate the function of a subset of these genes to better understand regulation and evolution of ommatidia development. In parallel we will combine state-of-the-art 3D imaging techniques, computational modelling, behavioural experiments and calcium imaging to explore the effect of genetic variation at the otd locus on insect vision thus fully linking genotype to phenotype. Taken together the results of this project will provide new insights into the regulation of eye development and organ size as well as the evolution of eye morphology and animal vision.

This proposal will have a beneficial impact on individuals and organisations in both private and public sectors through three main mechanisms. First, our research to understand eye genetics and development specifically and organ size regulation more generally will serve as a platform to potentially improve health and well-being. Second, through staff training and development, and capacity building. Third, through engagement with members of the public as well as children and teachers about the aims of our research, eye development and function, and the importance of understanding genetics, development and evolution.

Improving Health and Well Being
Investigating the regulation and development of eye size and how this evolves in Drosophila has great potential to help better understand the principles of eye development and organ growth more generally. In addition, the focal gene of this project, orthodenticle, is not only important for eye development and evolution, but its vertebrate homologue Otx2 underlies human eye diseases. Therefore, we will bring together academics and other relevant stakeholders, including representatives from biomedicine and publishing, at a workshop focusing on eye genetics, evolution, function and disease. This will facilitate the sharing of expertise on eyes and vision to catalyse new interdisciplinary research collaborations. Furthermore, dissemination of the outcomes of this event through the press will serve to increase public awareness of how basic research can help inform applied research as well as more specific aspects of research such as development and evolution.

Training and capacity building
The postdoc JH will develop further skills and expertise in wet lab biology but will also be trained in bioinformatics, bioimaging and analysis of animal behaviour. Providing this interdisciplinary training will result in a highly experienced researcher with the rare combination of wet lab and computational skills that will enable her to fully address important research questions. As well as continuing to enhance her scientific training, this project will provide JH with skills in leadership, grant management, supervision, mentoring and public engagement to furnish her with all of the necessary attributes for an independent research career following this project. In addition, she will disseminate the results of the project at prestigious international conferences thus enhancing the reputation of UK science.

The postdoc DW will similarly have access to training in leadership, grant management, supervision and mentoring as part of his staff development. In addition, since DW will work on this project on a part time basis while he further develops his own freelance research business, this arrangement will also help further align business development with biosciences and so potentially have an economic impact. To support this DW will have access to business development training here in Oxford Brookes University and be able to consult with researchers in our Bioinnovation Hub.

Increasing Public Engagement with Scientific Research
We will continue to build our public engagement programme to disseminate the aims and importance of our research to the general public. The outreach activities in this proposal will help children and teachers to better understand how genes interact to build animal eyes, how these eyes work and evolve to facilitate adaptations in vision. This proposal will also help us to engage with educators and the general public about the importance of genetics, development and evolution in their everyday lives and how basic research feeds that knowledge. This will complement our engagement with other stakeholders to improve scientific understanding and inform and develop education and science policies.