Steven G. Britt - US grants

The aim of the research proposed in this application is to gain insight into the molecular mechanism of signal transduction in the visual system. It is expected that these studies will contribute to a better understanding of the relationship between receptor structure, function, and information processing in biological systems. The genes encoding four Drosophila opsins have been isolated and characterized (Rh1-4). Each of these is expressed in distinct classes of photoreceptor cells and displays characteristic patterns of sensitivity to light. The Rh1 opsin is a blue sensitive pigment (lambda max 480 nm) which is expressed in the six outer photoreceptor cells (R1-R6). Rh2 displays light sensitivity with lambda max 420 nm and is expressed in the occelli. Rh3 and Rh4 are expressed in non-overlapping sets of UV sensitive R7 cells, and appear to correspond to the yellow and pale pigments expressed in these cells. In previous studies, a Drosophila mutant lacking the wildtype Rh1 gene product (NinaE) was transformed with a chimeric gene composed of the Rh1 promoter and the Rh2 structural gene. In these flies, the Rh2 opsin was misexpressed in R1-R6 cells and conferred new spectral and physiological characteristics to this class of photoreceptor cells. These studies demonstrate the feasibility of targeting gene constructs to specific photoreceptor cells for expression and analysis, and provides an ideal system for studying spectral tuning of Drosophila opsins in vivo. In an attempt to determine the molecular basis of spectral specificity and sensitivity of the visual pigment molecules we will: (1) Construct translational gene fusions between the different Drosophila opsins. (2) Express the chimeric opsins in the major class of photoreceptor cells of NinaE flies using P-element mediated germline transformation. (3) Characterize the spectral and physiological behavior of the transformants in vitro and in vivo in order to identify the regions or domains of the opsin molecule involved in spectral tuning. As these studies near completion, (4) We will begin the molecular characterization of other genes encoding proteins which appear to be involved in phototransduction.

The aim of the research proposed in this application is to gain insight into the molecular mechanisms of signal transduction in the visual system. It is expected that these studies will contribute to a better understanding of the relationship between receptor structure, function, and information processing in biological systems. The genes encoding four Drosophila opsins have been isolated and characterized (Rh1-4). Each of these is expressed in distinct classes of photoreceptor cells and displays characteristic patterns of sensitivity to light. The Rh1 opsin is a blue sensitive pigment (lambda/max 480nm) which is expressed in the six outer photoreceptor cells (R1-R6). Rh2 displays light sensitivity with lambda/max 420nm and is expressed in the occelli. Rh3 and Rh4 are UV sensitive and are expressed in non-overlapping sets of R7 cells. In previous studies, we generated a series of chimeric Drosophila opsin genes, in order to identify regions of the opsin protein that are involved in regulating rhodopsin sensitivity and metarhodopsin absorption. These chimeric proteins were derived from regions of the blue and violet sensitive pigments. The chimeric genes were expressed in the R1-R6 photoreceptor cells of ninaE mutant animals which lack the wild type Rh1 gene product. In these flies, the chimeric opsins are the only photopigments expressed in the R1-R6 photoreceptor cells. We examined the spectral sensitivity of animals expressing these modified pigments in vivo using physiological techniques, and studied the absorption of the activated form of the pigment (metarhodopsin) by in vivo microspectrophotometry. We found that multiple regions of the opsin protein are involved in regulating rhodopsin spectral sensitivity and that the native and activated forms of the pigment can be tuned independently. In the present application we propose to 1) identify regions of the opsin protein that are responsible for differences in the spectral sensitivity of the Drosophila Rh1 and Rh2 rhodopsins. 2) Identify residues in the second transmembrane segment of the opsin protein that are responsible for differences in the absorption of the activated form (metarhodopsin) of the Drosophila Rh1 and Rh2 rhodopsins. 3) Identify additional sites within the opsin protein that play an important role in rhodopsin sensitivity and function. 4) Isolate and characterize the genes encoding novel photopigments found in Drosophila and other invertebrates. 5) Generate and characterize transgenic flies that ectopically express opsins from other invertebrate and vertebrate organisms. These studies will provide insight into the mechanisms underlying the regulation of rhodopsin spectral sensitivity and the relationship between rhodopsin structure and the process of photoactivation. These issues are central to the study of vision, and will provide a unique model system for understanding how G-protein coupled receptors function.

The overall objective of this research proposal is to determine the molecular mechanisms by which photoreceptor cell fate is specified. This will be accomplished by using a combination of molecular biological and genetic techniques. The system of study will be the compound eye of the fruit fly Drosophila melanogaster. In recently published work, we identified and characterized a novel pattern of visual pigment gene expression in the fly eye. By manipulating the eye genetically we have shown that the expression of visual pigment genes in one photoreceptor cell type. It appears that one cell in each ommatidium (R7) adopts one of two different cell fates in a stochastic manner, and then communicates this decision (inductively) to the adjacent R8 cell. These events serve to coordinate the expression of visual pigments in these two cells, and produce two type of optical units within the eye that have distinct spectral sensitivities. In this proposal, we present a series of experiments to rigorously test this model, by examining aspects of opsin gene expression, studying the promoters which regulate their expression, and conducting genetic screens to identify genes which are required for the specification of photoreceptor cell fate and the regulation of opsin gene expression. These studies are of fundamental importance in understanding how the compound eye is formed. The differentiation of photoreceptors into populations having different spectral sensitivities is the basis for color discrimination. Signal trafficking within the nervous as a whole is also dependent upon the generation f diverse neuronal populations having different sensitivities and transmitter phenotypes. Because the fly eye has become a key model system in Developmental Biology, the elucidation of novel signaling and cell-fate determination pathways within this system will serve as a framework to understand more complex developmental events within the nervous system of other organisms.

Color vision in butterflies is dependent upon the presence of different photoreceptor neurons within the retina, which are sensitive to different colors of light. Just as in many vertebrate and invertebrate species, this results from the highly regulated expression of different forms of the light sensitive visual pigment rhodopsin, in different types of photoreceptor cells. During evolution, gene duplication events have produced numerous forms of rhodopsin in different species. The PIs will study this evolutionary process and the origin of the different forms of rhodopsin. The PIs will examine the rhodopsins of the Tiger Swallowtail butterfly (Papilio glaucus) as well as those of more distantly related species. The PIs will measure the colors of light absorbed by the different butterfly rhodopsins and determine which specific photoreceptor cells within the retina express each form of rhodopsin. The PIs will also compare the amino acid and nucleotide sequences of these and other rhodopsins to determine whether the rhodopsins absorbing the same wavelengths of light are likely to have evolved from a common ancestral gene or if they may have evolved independently. These studies will clarify the evolutionary mechanisms by which color vision was established, and contribute to the basic knowledge of the visual system of insects.

Award Abstract

The overall aim of this project is to better understand color vision in insects and other invertebrate animals. Insects use color as an important cue in navigation, avoiding predators and identifying food sources. Dr. Britt will study how different forms of the visual pigment rhodopsin are tuned to different colors of light. Using the fruit fly Drosophila melanogaster as a model system, Dr. Britt's laboratory will identify specific amino acids within the rhodopsin protein that are responsible for differences in color sensitivity and that play a role in activating the protein when light is absorbed.
Drosophila melanogaster utilizes forms of rhodopsin that have unique color properties, and these visual pigments can be studied using well-developed molecular biological, genetic and physiological methods. In addition, this experimental system provides the opportunity to examine diverse invertebrate visual pigments, such as those from the butterfly Papilio glaucus. Dr Britt will conduct comparative experiments between fruit flies and butterflies with the aim of identifying fundamental principles underlying the similarities and differences in how these animals distinguish between different colors of light. Dr. Britt's work may ultimately provide important insight into how light influences insect behavior, and thus a means to manipulate insect behavior. Dr. Britt is also an active mentor for students of underrepresented minorities and involves them in his research.

DESCRIPTION (provided by applicant): Cell-fate specification plays an essential role in the ultimate function of the nervous system. Generation of diverse cell populations and the regulation of their precise placement and connectivity patterns establishes neural networks capable of detecting, processing and sending complex signals. Cell identity, position and connectivity are especially important in sensory systems because of the added complexity of spatial information that must be detected and encoded. High-resolution sampling of visual space by the retina demands a dense array of photoreceptor cells sensitive to a wide dynamic range of light intensities. Moreover, color vision requires photoreceptor cells having different spectral sensitivities in addition to a precise retinotopic map. Currently, little is known about the generation of photoreceptor cell diversity or the specification of different spectral types. Drosophila melanogaster is capable of color vision and is a useful experimental system for examining the developmental programs that produce photoreceptor cells having different color sensitivities. We have found that a very specific inductive signal between adjacent photoreceptor cells coordinates their fates and color sensitivities. We have identified a large group of genes that influence this inductive signal. These genes include members of the Epidermal Growth Factor Receptor, nephrin related immunoglobulin superfamily (IgSF), and Notch signaling pathways. The aim of this proposal is to determine how the individual members of these signal transduction pathways function to establish photoreceptor cell-type adjacency and pairing, and to examine how these pathways interact in a coordinated way to regulate the inductive signal between adjacent photoreceptors. This work will provide a better understanding for how specific developmental signals operate during eye development and establish the patterned photoreceptor cell mosaic that is capable of color vision. PUBLIC HEALTH RELEVANCE: In our previous work, we have identified a group of genes that are required to establish the precise cell-cell adjacency of the R7 and R8 cell types in pale and yellow ommatidia. The purpose of this proposal is to define how each of the individual genes function in this process and to determine how they interact with each other overall. Because the molecular mechanisms that regulate eye development in different organisms are highly conserved, we believe that using Drosophila melanogaster as a model system to identify and characterize the genes responsible for photoreceptor cell patterning will provide important information that will be relevant to retina development in general. Furthermore, we believe that our analyses of the integration of multiple signal transduction pathways will also provide important insights that will be relevant to a variety of developmental and signaling processes in both health and disease.

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how stochastic cell fate determination regulates the formation of the retinal mosaic. Continued existence of this gap represents an important problem in retinal development because, until it is filled, the understanding of normal and pathological eye development will remain fragmented and incomplete; The long-term goal is to understand how different classes of photoreceptors are specified by stochastic mechanisms that are intrinsic to an individual photoreceptor. The objective in this particular application is to identify additional genes involved in regulating the stochastic determination of R7 photoreceptor cell fate in an effort to develop a coherent gene network that integrates the function of spineless and tango into a comprehensive testable model. The central hypothesis is that a complex gene network controls the specification of Rh3 and Rh4 expressing R7 photoreceptor cells that includes spineless, tango, as well as additional unidentified genes. This hypothesis has been formed based on preliminary data produced in the applicant's laboratory. The rationale for the proposed research is the observation that the proportion of R7 photoreceptors that express one visual pigment or another is a continuously variable quantitative trait. This suggests that the genes within this network, which produces these different photoreceptor cell types, can be identified and characterized using quantitative genetic approaches. Guided by this strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify Quantitative Trait Loci that regulate R7 photoreceptor cell fate and opsin expression; and 2) Develop and test a gene network model based on these Quantitative Trait Loci. Under the first aim, a proven quantitative genetics approach will be applied to examine the specification of Rh3 and Rh4 expressing R7 photoreceptor cells. Under the second aim, the identified genes will be assembled into a network model and tested for their ability to regulate R7 photoreceptor cell subtype specification. The approach is innovative, because it uses new technologies to examine the mechanics of retinal development in a way that has not been previously undertaken and because this approach is likely to provide a type of information that promises to fundamentally alter our understanding of eye and retina development. The proposed research is significant, because it is expected to vertically advance and expand the understanding of how the retina is formed and develops. Ultimately, such knowledge has the potential to impact the understanding and treatment of congenital eye defects and malformations and influence the development of new treatments for visual system diseases through the manipulation of specific cells within the retina.

PROJECT SUMMARY ABSTRACT There is a fundamental gap in understanding the etiology of retinal disorders, such as retinitis pigmentosa and age-related macular degeneration. This gap in our understanding is compounded by complex differences in the underlying genetics, variation in individual disease severity, and the effects of the environment. The continued existence of this gap represents an important problem in retinal disorders because, until it is filled, the under- standing and treatment of these eye pathologies will remain fragmented and incomplete. The long-term goal is to understand how different genes and the variation in their expression contribute to visual system impairment. The objective of this particular application is to characterize the transcriptional profile of a panel of highly inbred fruit fly strains (the Drosophila Genetic Reference Panel, DGRP) and study the relationship between gene ex- pression, genomic polymorphisms and the substantial variation in visual system function and age-related visual impairment within the DGRP. The central hypothesis is that a complex genetic network composed of gene co- expression modules is responsible for visual impairment in these strains. This hypothesis has been formed based on published work and preliminary data produced in the applicant's laboratory. The rationale for the proposed research is the observation that multiple genetic polymorphisms have been identified in Genome Wide Associa- tion Studies of visual system impairment in the DGRP. However, recent studies have failed to identify a tran- scriptional link between these genetic polymorphisms and the impairment of vision. Using a Systems Genetics approach, our hypothesis will be tested by pursuing two specific aims: We will 1) Identify Genetic Co-Expression Networks that regulate visual system function and impairment, and 2) Develop and test gene network models based on these candidate co-expression modules and candidate genes. The approach is innovative because we will examine the interactions of genotype, transcriptome and visual system function using a highly statistically powered Systems Genetics approach incorporating a tissue focused Tag-Seq transcriptome analysis. This ap- proach is likely to provide a type of information that promises to fundamentally alter our understanding of the molecular genetics of visual system function and disease. The proposed research is significant because it will vertically advance our understanding of the genome-transcriptome relationship and how it impacts the eye. Be- cause many of the genes under study are associated with human retinal dystrophies, the fundamental knowledge from the project has the potential to advance the understanding and treatment of visual system disease.