James M. Fadool - US grants

DESCRIPTION (Adapted from applicant's abstract): The long-term objective of the proposed research is to uncover genetic and molecular mechanisms of cell differentiation in the vertebrate retina. Recessive mutations affecting the development of the eye in zebrafish (Danio rerio) have been recovered from a chemical mutagenesis screen, and the applicant proposes to further characterize these mutant lines. The first aim is to examine the mutant eyes by histological and immunological analysis, and to select a few lines for further analysis. The second aim is to define the nature of the mutated genes by genetic mosaic analysis (transplanting cells from mutant into wildtype embryos) and by complementation analysis (searching for additional alleles of the mutated genes by genetic methods).

DESCRIPTION (Adapted from the applicant's abstract): A high-throughput enhancer trap technique will be refined and subsequently exploited to systematically define hundreds of genes that are important in the retinal development of zebrafish. The technique is a powerful adaptation of well-characterized P-transposon methods that have been so effective in dissecting the genetics of Drosophila development. In this case, the method relies on the selective expression of transposons tagged with a nontoxic fluorochrome, i.e., green fluorescent protein (GFP). Relatively efficient integration of the transposon has been demonstrated, and more than 5 percent of integration events are expected to lead to GFP expression under the control of endogenous enhancers in select populations of cells and tissues. Lines of fish in which GFP is expressed selectively in retina or other tissues will be identified and preserved for more detailed developmental, morphometric and genetic analyses by the principal investigator and others. As in Drosophila, a significant fraction of transposition events is expected to disturb normal gene expression and to lead to mutations or quantitative variants. This feature of the enhancer trap is particularly significant because it should enable a fine-grained analysis of subtle abnormalities in retina that would otherwise escape detection.

DESCRIPTION (Adapted from the applicant's abstract): A high-throughput enhancer trap technique will be refined and subsequently exploited to systematically define hundreds of genes that are important in the retinal development of zebrafish. The technique is a powerful adaptation of well-characterized P-transposon methods that have been so effective in dissecting the genetics of Drosophila development. In this case, the method relies on the selective expression of transposons tagged with a nontoxic fluorochrome, i.e., green fluorescent protein (GFP). Relatively efficient integration of the transposon has been demonstrated, and more than 5 percent of integration events are expected to lead to GFP expression under the control of endogenous enhancers in select populations of cells and tissues. Lines of fish in which GFP is expressed selectively in retina or other tissues will be identified and preserved for more detailed developmental, morphometric and genetic analyses by the principal investigator and others. As in Drosophila, a significant fraction of transposition events is expected to disturb normal gene expression and to lead to mutations or quantitative variants. This feature of the enhancer trap is particularly significant because it should enable a fine-grained analysis of subtle abnormalities in retina that would otherwise escape detection.

Males of many species possess extravagant, brightly colored structures that are costly to produce and that increase their likelihood of being seen and eaten by predators. One of the great problems of evolutionary biology is understanding why females select for such extravagant traits in males. The "sensory bias hypothesis" states that female mating preferences are by-products of natural selection on the sensory system. Under this hypothesis, strong natural selection on animals to find food, avoid predators, and find proper habitats results in indirect selection on mating preferences. The fundamental assumption of this hypothesis is that the genes (and neurological pathways) that affect foraging preferences also affect mating preferences. This project will determine the extent to which different behaviors that share a common sensory system can evolve independently. The objective of the study will be to determine if behaviors share a common sensory system and/or does variation in the sensory system lead to variation in behavior. To address these questions, large amounts of phenotypic variation in visual properties will be generated and multiple aspects of behavior will be measured using the bluefin killifish, Lucania goodei, a species previously demonstrated to have large amounts of variation in visual properties both within and between populations.
Broader Impacts: The study will provide the graduate student and several undergraduates experience with a variety of techniques drawn from many disciplines. This project involves the collaboration of evolutionary biologists and molecular biologists/vision physiologists that will help build connections between the evolutionary biology group within the Biological Science Department and other groups, particularly the university-wide Program in Neuroscience. The project should demonstrate that studying natural variation in vision physiology is a worthwhile endeavor, while developing further insights into the evolution of vision physiology.

DESCRIPTION (provided by applicant): The vertebrate eye and retina have provided important models to identify fundamental processes of developmental such as induction, morphogenesis, patterning and cell fate specification. While a considerate amount of information describing the early events of eye development has been uncovered, many processes of later stages of differentiation and maturation are less well understood. For example, the well characterized, laminar organization of the vertebrate retina is complimented by the non-random or mosaic arrangement of neurons within each of the layers. Though the necessity of the mosaic arrangement is intuitive: gaps in the distribution of neurons or clustering of cells would result in under representation or oversampling of portions of the visual field, little is known of the genetic mechanisms regulating the mosaic patterning. Over the next five years, we propose a novel genetic screen of post-embryonic stage larvae and adult progeny of ENU-mutagenized zebrafish to uncover novel recessive and dominant mutations affecting the visual system. A systematic screen for late onset mutations should provide much needed models of inherited diseases in humans such as retinitis pigmentosa, congenital cataracts and glaucoma. Three specific aims are proposed: SPECIFIC AIM I: Recover novel, recessive mutations that specifically affect the specification and mosaic patterning of photoreceptor cells through an in situ immunohistochemical screen of the rod mosaic in free swimming, 5 day old larvae. SPECIFIC AIM II: Identify genes essential to the development and maintenance of the anterior segment, through a morphological screen for defects in the lens, cornea and pupil of the larval eye. SPECIFIC AIM III: Identify mutations resulting in photoreceptor cell dystrophies as models of human retinal disease through the histological and immunofluorescent analysis of retinas of adult zebrafish. The initial mutagenesis procedure and subsequent breeding strategy incorporated a mapping panel into the mutagenized lines to facilitate more efficient mapping and the subsequent cloning of the mutated genes. Descriptions and images of the mutant phenotypes will be available at the PI's website and the Zebrafish Information Network and distribution will be handled through the Zebrafish International Resource Center.

? DESCRIPTION (provided by applicant): Our research is driven by the prospect that the progressive nature of photoreceptor degeneration leaves open an opportunity for treatments that delay the time-course of cellular damage and forestall vision loss. Humans are largely dependent upon cone-mediated central vision. However, in a significant number of people, death or dysfunction of rods results in the secondary loss of cones, remodeling of retinal circuitry and blindness. The heterogeneous nature of the molecular defects underlying retinitis pigmentosa (RP) has made identifying the specific mechanisms leading to degeneration difficult. Even less understood, are the principal causes of the secondary death of photoreceptors not expressing the mutated gene. There exists a critical need for robust models that recapitulate the pathological sequence leading to disease and to rapidly and reliably screen for agents to lessen the impact of the disease. My laboratory has taken advantage of the genetic manipulations available in zebrafish to identify genes regulating photoreceptor development and survival. In this proposal, we will directly address two common features observed in people affected by a wide range of photoreceptor disease; the cellular alterations that precipitating initial photoreceptor death, and secondary alterations leading to blindness. We propose a two prong approach: 1) Apply innovative genetic manipulations to introduce analogous mutations in zebrafish rhodopsin as precisely defined models of human retinal disease; 2) Use these and our existing degeneration models to screen for small molecules that modify disease progression. Completion of the specific aims will significantly advance the field's understanding of alterations underlying photoreceptor death, and identify potential novel targets or therapeutic agents as lead compounds to confer protection from the secondary consequences of photoreceptor degeneration.

PROJECT SUMMARY Despite our dependency upon cone-mediated vision, and the debilitating impact of cone degeneration upon our central vision, major gaps exist in our knowledge of genes and mechanisms that regulate the number and spatial patterning of photoreceptors. Underlying this asymmetry is the fundamental choice of retinal neuroblasts to re-enter mitosis or differentiate. The larval zebrafish retina provides an unparalleled genetic model to identify fundamental mechanisms integrating photoreceptor specification and spatial patterning. The larval zebrafish retina is anatomically and functionally cone-dominated with 4 cone subtypes arranged in a highly ordered mosaic. Rods are far less numerous and distributed asymmetrically along the dorsal/ventral axis. The range of genetic tools, availability of genomic resources and access to the zebrafish embryo, permit a systematic approach to uncover the impact of genetic manipulations upon cell fate and their distribution. Our premise is based upon rigorous genetic and molecular analysis in our published studies and preliminary data showing that tbx2b/lor and six7/ljr are essential for maintaining the cone-dominated zebrafish retina. Mutations of tbx2b result in a 5-fold increase and uniform distribution of rods due to a cell fate switch of SWS1- cones into rods, a phenotype opposite to that of the rd7 mouse and enhanced-S cone syndrome in humans. Mutations of six7, an orthologue of Six3/6 result in a similar increase and uniform distribution of rods, however characterization of several alleles revealed that six7 independently suppresses mitosis of late stage progenitors, and is essential for survival of a cone subtype. Our goal is to understand the mechanisms underlying functions of tbx2b and six7 in cell fate decisions and spatial patterning in the context of know factors that regulate photoreceptor development. Aim 1 will take advantage of innovative genome editing tools, transposon-based transgenesis, and more routine methods to test the hypothesis that six7 modulates the choice of late stage photoreceptor progenitors between continued mitosis or differentiation. Aim 2 combined in vivo and in vitro approaches to test our novel hypothesis that tbx2b regulates the timing of photoreceptor determination and thereby spatial patterning through molecular interaction with known photoreceptor transcription factors. Furthermore, our unpublished data and published reports show unexpected alterations in gene expression following genetic alterations suggesting unrecognized roles for these factors in maintaining cell fate. Therefore, completion of the specific aims will significantly advance the fields knowledge of mechanisms regulating the generation of a highly specialized, cone-dominated retina.