Herwig Baier - US grants

We propose to study visual processing in a novel manner, by isolating zebrafish mutants on a large scale. In particular, we are interested in the segregation of visual functions in the brain, a well-known but poorly-understood feature of vertebrate vision. Different kinds of visual information are processed by highly specialized neurons in the retina and channeled into separate brain nuclei. These pathways in turn generate distinct behavior patterns in response to visual stimulation. We propose to establish a functional map of the zebrafish visual brain by using genetic lesions as a dissecting tool. Six different behavioral assays will be used to search for mutations affecting specific visual functions in a screen modeled after the highly successful screen for mutants in the retinotectal projection. We will test for optomotor responses and optokinetic responses to moving gratings, and for adaptation of skin pigment to ambient light levels (a process requiring retinal input). The optomotor assay will be combined with two motion- nulling paradigms (borrowed from human psychophysics), which test for the intactness of color and luminance channels. Our pilot screens predict that this large-scale approach will uncover a broad assortment of several hundred mutants. The specificity of the phenotype will vary with the site and extent of the genetic lesion. Many phenotypes will allow us to assign functions to certain nuclei, pathways, or cell types. Some mutants will remain puzzling, particularly those with no detectable anatomical disruption. As a tool for the screen, mutagenized males will be crossed into a line of transgenic fish, which express green fluorescent protein in retinal axons. This genetic background will later permit the rapid detection of mutations in retinorecipient areas. In parallel to the mutant screen, we will investigate the types of retinal ganglion cells and their axonal projections by DiI tracing, both in wildtype and in selected mutants. Health-relatedness. Given the large degree of conservation of genes between fish and humans, this approach directly addresses the problem of inherited human blindness, and leads a way to isolating the genes involved in diseases of the human retina and central visual pathways.

[unreadable] DESCRIPTION (provided by applicant): The molecular mechanisms underlying visual-system development are only beginning to be understood. The long-term objectives of this work are the identification and characterization of molecules that govern the differentiation of neuronal classes in the visual pathway. The vertebrate retina is composed of over 50 neuronal cell types that fall into 6 major classes. This proposal focuses on one prominent class, the retinal ganglion cells, which connect the eye to the brain. The current model of retinal neurogenesis proposes that cell classes are generated during development from multipotential progenitors in an invariant temporal order. Ganglion cells are always born first; they emerge from a sheet of undifferentiated progenitor cells in the form of a wave that sweeps across the embryonic eye. In this grant, we will use a genetic approach in zebrafish, to investigate the molecular interactions that underlie the ganglion-cell differentiation wave. The recently discovered transcription factor Ath5 is of central importance, since its mutation in the zebrafish mutant lakritz eliminates all ganglion cells. We will test its interactions with the factors Shh, Twhh, Fgfl, and Brn3c, which have been implicated in ganglion-cell genesis. We will further ask whether Ath5 is also involved in subsequent steps of differentiation of retinal ganglion cells. Finally, we will attempt to discover novel genes in a mutagenesis screen taking advantage of a transgenic zebrafish line that expresses GFP in ganglion cells. These studies will establish a genetic pathway of factors involved in determination, differentiation, and diversification of retinal ganglion cells. Health-relatedness. This approach may lead a way to isolating genes involved in diseases of the human retina, such as glaucoma and optic-nerve hypoplasia, for which there are currently no cures

We propose to study visual processing in a novel manner, by isolating zebrafish mutants on a large scale. In particular, we are interested in the segregation of visual functions in the brain, a well-known but poorly-understood feature of vertebrate vision. Different kinds of visual information are processed by highly specialized neurons in the retina and channeled into separate brain nuclei. These pathways in turn generate distinct behavior patterns in response to visual stimulation. We propose to establish a functional map of the zebrafish visual brain by using genetic lesions as a dissecting tool. Six different behavioral assays will be used to search for mutations affecting specific visual functions in a screen modeled after the highly successful screen for mutants in the retinotectal projection. We will test for optomotor responses and optokinetic responses to moving gratings, and for adaptation of skin pigment to ambient light levels (a process requiring retinal input). The optomotor assay will be combined with two motion- nulling paradigms (borrowed from human psychophysics), which test for the intactness of color and luminance channels. Our pilot screens predict that this large-scale approach will uncover a broad assortment of several hundred mutants. The specificity of the phenotype will vary with the site and extent of the genetic lesion. Many phenotypes will allow us to assign functions to certain nuclei, pathways, or cell types. Some mutants will remain puzzling, particularly those with no detectable anatomical disruption. As a tool for the screen, mutagenized males will be crossed into a line of transgenic fish, which express green fluorescent protein in retinal axons. This genetic background will later permit the rapid detection of mutations in retinorecipient areas. In parallel to the mutant screen, we will investigate the types of retinal ganglion cells and their axonal projections by DiI tracing, both in wildtype and in selected mutants. Health-relatedness. Given the large degree of conservation of genes between fish and humans, this approach directly addresses the problem of inherited human blindness, and leads a way to isolating the genes involved in diseases of the human retina and central visual pathways.

DESCRIPTION (provided by applicant): The long-term goal of this work is to identify the genetic mechanisms underpinning the development and function of the visual system in a systematic and unbiased fashion. In the period preceding this grant proposal, we have carried out forward-genetic screens in search of mutations that disrupt visual functions of zebrafish. We introduced new mutations at random positions in the genome using the chemical mutagen ethylnitrosourea. Semi-automated behavioral screening assays were devised and employed to detect deficits in visual processing in over 2,000 mutagenized families. We also screened over 400 existing zebrafish mutants. This effort led to the identification of over 80 mutants. In this grant, we will now study the orderly formation of retinotectal synapses using two of the mutants discovered in our screen. We are particularly interested in the role of axon-axon interactions and activity-dependent synaptic competition in determining the exact position and territory that an individual retinal arbor is occupying in the tectum. One of our mutants lacks all retinal ganglion cells -- its tectum is genetically depleted of visual input. We will now transplant single or few wildtype ganglion cells into this mutant and ask if the position and shape of the axon arbor is altered when no other retinal axons are present. Another mutant has altered synaptic transmission properties allowing us to ask what role electrical activity plays in shaping retinotectal connections and how an activity-deficient axon arbor develops when it is surrounded by axons with normal activity levels. Finally, we propose to generate transgenic lines that allow us to conditionally block synaptic transmission from ganglion cells to neurons in the tectum. Transplantations and single-axon labelings will again serve to reveal the role of activity and synaptic competition in the establishment and stabilization of neuronal connections. Health-relatedness. Human and fish visual systems are very similar. Our approach, therefore, may lead a way to understanding the molecular basis of inherited and acquired afflictions of the visual pathway in human patients.

DESCRIPTION (provided by applicant): Synapses between cells with similar response properties are often clustered in space and segregated from synapses with different characteristics, giving rise to a layered (or laminar) organization. Little is known about the cellular and molecular mechanisms underlying the precise laminar targeting of axons and dendrites in any brain area. We are using a functional genetic approach in zebrafish to address this question. The inner plexiform layer of the zebrafish retina provides an experimentally tractable system for the analysis of synaptic lamination. Here axons of bipolar and amacrine cell axons form synapses on dendrites of retinal ganglion cells in about ten sublaminae, whose combined synaptic activity represents the visual image. Another important example of laminar architecture is found in the optic tectum. Here axons of individual retinal ganglion cells terminate in only one of four retinorecipient layers. Because we have so far found no evidence in zebrafish that visual experience plays a role in patterning retinal or tectal lamination, our working hypothesis is that these two stratification events are genetically hardwired. Consistent with this hypothesis, we have already identified in forward-genetic screens three zebrafish mutants, moonraker(mra), notorious (noto), and dragnet (drg), each with unique lamination defects in the retina, in the tectum, or in both areas. Using these mutants, we propose to investigate three or possibly four potential mechanisms by which retinal ganglion cells select the correct lamina in which to place their synapses. Aim 1 will ask if homotypic cell-cell interactions are required for laminar targeting of dendrites and axons. Aim 2 will test the role of cholinergic amacrine cells in providing a laminar scaffold for ganglion cell dendrites. Aim 3 will define the exact role of an extracellular matrix component, which we have already identified by positional cloning, in an axon's lamina choice. Aim 4, finally, proposes to identify a novel gene with unknown activity, but strikingly specific mutant phenotype in axon and dendrite lamination. Health-relatedness. Loss of retinal ganglion cells in human patients, through injury or disease, has devastating and currently irreversible consequences for vision. In the future, any therapeutic attempt to stimulate regeneration will not only have to replenish ganglion cells, but also facilitate their integration into the existing synaptic circuitry. This study proposes to identify the molecules that are important for this process during development and growth of the vertebrate organism, thus.

DESCRIPTION (provided by applicant): In this application, we propose to develop genetic tools for the conditional, reversible, and tissue-specific silencing of neuronal activity in zebrafish. We then intend to use these new tools to carry out an enhancer trap screen with the aim of characterizing the zebrafish central nervous system in terms of its connectivity and functional architecture. This program will initially entail the use of the bipartite GAL4/UAS system, driving a chemically gated chloride channel in subsets of neurons. This will allow both spatial control (using tissue specific promoters) and temporal control (based on the presence or absence of the chemical gate) over protein function. A major goal of this proposal is to develop and characterize these new genetic techniques and to make the reagents available to the research community. With the ability to silence neurons conditionally and reversibly, we will then undertake an enhancer trap screen, aiming to identify a variety of expression patterns in the central nervous system. An early goal of this screen will be to characterize the expression patterns in terms of their cellular compositions, the fine structures of their individual neurons, and the pre- and postsynaptic connections that the neurons make. This preliminary work will provide important information about the connectivity within and among distinct structures in the zebrafish brain. With genetic access to these tissues, we will then silence neurons composing the trapped expression patterns, and perform behavioral analyses of the affected fish. By systematically assaying for a variety of behaviors in fish with a wide variety of expression patterns, we will eventually develop a functional atlas of the zebrafish brain, relating neural circuits to the behaviors that they mediate. Various tissues and cell types involved in the same behaviors will likely be connected functionally, and we will investigate such possible relationships anatomically. Health-relatedness: The connectivity of the human and fish nervous systems are very similar. By functionally characterizing the circuitry of the zebrafish brain, we also hope to provide insights into the pathophysiology of human behavior. The reagents and techniques developed under this proposal will advance biomedical research throughout the zebrafish community.