Steven C. McLoon - US grants

Fetal retina transplanted to the midbrain region of a newborn or adult rat survives, differentiates and forms connections with appropriate visual nuclei in the host brain. The project proposed here will examine further the development of retinal transplants in order to gain more insight into mechanisms active during development of normal retinal as well as further assess the potential for the use of retinal transplants as a clinical technique. This project has three major parts. First will be to evaluate possible mechanisms by which axons from retinal transplants find the appropriate visual nuclei. Particular attention will be given to the possibilities that transplant axons may initially be broadly distributed and then selectively retain only appropriate projections, that transplants axons may only follow host optic axons, or that transplant axons may preferentially tend to grow along the surface of the brain. This will involve using neuroanatomical techniques to study the developing projection of retinal transplants in normal and anophthalmic rats and transplants placed in various locations in the host brain. Second will be to determine if transplants of fetal retina made to the orbit can survive and form connections with the host brain. Third will be to evaluate the role of glia in directing the migration of neuroblasts in the developing retinal transplants. This will involve using immunohistochemical techniques with antibodies specific for retinal glia to identify the origin and distribution of glial cells in the developing transplants and to attempt to produce retinal transplants devoid of glia. This project will provide a greater understanding of mechanisms active in the devlopment of the normal visual system. With this understanding we can begin to construct the parameters required for using retinal transplants as a therapeutic means for treating retinal blindness.

The ganglion cells of the mature retina project to the visual nuclei of the brain in a highly organized fashion. We have shown that during early development these projections are much less organized. This study will further characterize the optic projections in the immature visual system and investigate possible developmental mechanisms for the establishment and refinement of these early projections. There are five major lines of investigation. First, the degree of order between growing optic axons in the optic nerve, tract and tectum will be examined. This is to determine if there is sufficient order between ingrowing axons to account for the initial order in the terminal field. Second, the retinal ganglion cells giving rise to the earliest tectal projection will be identified. This is to evaluate the role fo temporal gradients in orienting the retinal map on the tectum. Third, timing will again be evaluated as a mechanism for determining the laminar distribution of ganglion cell axons in the tectum. This will involve determining the time and pattern of ingrowth of axons from the different ganglion cell classes to the tectum. Fourth, we will determine whether cell death is responsible for removing aberrantly projecting ganglion cells from the developing retina or if these cells can retract their aberrant collaterals. This will require labeling aberrantly projecting cells early in development and determining whether these labeled cells survive through the development of the visual system. Fifth, we will further characterize the role of the central visual nuclei in developmental cell death in the retina. This will involve attempts to alter the cell death in the ganglion cell layer by increasing the size of the tectum and attempts to demonstrate a specific trophic action of the tectum on retina.

[unreadable] DESCRIPTION (provided by applicant): A group of 15 vision scientists at the University of Minnesota request continued support for training up to seven predoctoral students a year. Students come from the Graduate Program in Neuroscience or a related field with a minor in neuroscience. Trainees eligible for support have completed at least their first year of graduate school and have begun their thesis research in a laboratory of one of the vision training faculty. Trainees earn a Ph.D. or M.D./PhD. and can receive support through the duration of their thesis research, typically less than four years. The Neuroscience Program at the University of Minnesota is uniquely positioned to provide broad training in neuroscience, together with specialized training in areas related to vision. The vision research community at the University is growing and currently includes many highly respected investigators in the field. The diverse research interests of this group of scientists range from molecular biology to visual perception. This group, coupled with the many other nationally recognized neuroscientists at the University of Minnesota, makes this a natural place to center a training program in the neuroscience of vision. The training program led by those investigators is preparing vision scientists with the multi-disciplinary education needed to meet the challenges of the future. Relevance: The retina and central visual pathways share many common features in terms of development, organization and function with the rest of the nervous system. The visual system also shares in the spectrum of disorders found in other parts of the nervous system, and common themes may eventually explain seemingly diverse disease states ultimately leading to common cures. This points to a critical need for training future vision scientists with a knowledge of the nervous system. [unreadable] [unreadable]

DESCRIPTION: (Applicant's Abstract) A growing body of work suggests that retinal cells carry positional markers. These markers serve to distinguish cells in different regions of the retina from one another, and they appear to be important in mediating specific cell-cell interactions. Although there is strong evidence for functional differences in the cells on the nasal and temporal sides of the developing retina, there is limited knowledge of the molecular diversity that underlies these differences. The goal of this project is to identify molecules asymmetrically expressed in the nasal-temporal axis of the developing retina and to determine the function of these molecules. The project is divided into four specific aims.First, TRAP, a molecule expressed by most ganglion cells on the temporal side of the retina and by few ganglion cells on the nasal side, will be characterized in more detail. This will involve cloning the gene for TRAP from a cDNA library, sequencing the gene, and developing antibodies to the protein for use in analyzing the function of TRAP. Second, three complementary approaches will be used to identify other molecules expressed asymmetrically between the nasal and temporal sides of the developing retina. These approaches include the use of monoclonal antibodies, cDNA cloning by subtractive hybridization, and lectin blots. As molecules are discovered, they will be characterized as described for TRAP. Third, anatomical differences between the nasal and temporal sides of the retina will be identified.This information will be used to develop in vivo assays to study the function of side-specific molecules and will allow specific patterns of axon growth to be correlated with the distribution of asymmetrically distributed molecules. Fourth, the function of molecules asymmetrically expressed in the developing retina will be examined. This will involve perturbing the function of these molecules with antibodies in vivo and in vitro. Preliminary studies will also evaluate techniques for introducing genes to alter the expression of specific molecules.

DESCRIPTION (from applicant's abstract): Axons of retinal ganglion cells connect to cells in the visual centers of the brain in a precise, stereotypical pattern. This pattern of connections is essential for normal visual function. The goal of this project is to further our understanding of how the proper pattern of connections develops between retinal axons and the central visual centers. This project is guided by the hypothesis that the pattern of retinal connections in the brain is established in part due to a set of cytochemical positional labels carried by the retinal axons and by cells in the target centers to which the axons connect. This project is a continuing effort by this laboratory to identify these positional labels. Available evidence suggests that gradients of specific Eph receptor tyrosine kinases expressed across the retina could be involved in detecting positional labels in the central visual centers. The role of these receptors in development of the visual system has never been directly tested. The overall aim of this project is to determine the role of members of the Eph subfamily of receptor tyrosine kinases in the development of the normal pattern of retinotectal connections. The general approach to this project is to alter the expression by retinal cells of specific receptors and then study the resulting pattern of retinotectal connections in developing chick embryos. The first specific aim is to reduce expression of Cek4, an Eph receptor tyrosine kinase, in the developing retina, and then characterize the effect this has on the pattern of retinotectal connections. Cek4 is normally expressed in a nasal-temporal gradient in the developing retina. Antisense oligonucleotides to Cek4 or control sequences will be injected into the developing retina. The second specific aim is to misexpress Cek4 on the nasal side of the developing retina and then characterize the effect this has on the pattern of retinotectal connections. Retroviruses carrying the Cek4 gene or control sequences will be injected into the nasal side of the developing retina. The third specific aim is to determine if altered expression of Cek4 in the retina changes the nature of the interactions between retinal axons and tectal neurons or nonneuronal cells. The interaction between axons and tectal cells will be observed in tissue culture. The fourth specific aim is to study the effect of altering expression of other Eph receptor protein tyrosine kinases on the pattern of retinotectal connections using approaches similar to those described for Cek4. Initial work will focus on Cek5, which is expressed in a dorsal-ventral gradient in the developing retina.

DESCRIPTION (provided by applicant): Degeneration of retinal photoreceptor neurons, such as that seen in age-related macular degeneration (AMD), is the most common cause of blindness in the United States. There are compelling reasons to believe that subretinal cell transplantation could be used to replace missing photoreceptor neurons. No effective and practical source of cells for transplantation is currently available. The goal of this project is to develop cells to be used for transplantation to replace photoreceptor neurons in AMD and related diseases. Bone marrow-derived stem cells offer numerous advantages over other cell types as a possible source of donor cells. These cells can differentiate into neurons. They are readily available, and if used for autologous transplantation to the retina, they would not have the same immunological consequences inherent in the use of other cell types. To our knowledge, no other laboratories are investigating bone marrow-derived stem cells for transplantation to the retina. At this time, there is no evidence that bone marrow-derived stem cells can differentiate into retinal neurons. The specific aim of this preliminary investigation is to determine conditions that would allow these cells to differentiate as photoreceptor neurons or other retinal cell types. The study has three sequential steps. First, treat GFP-labeled, bone marrow-derived stem cells in ways likely to induce the photoreceptor phenotype. This includes culturing cells in factors such as FGF-2, EGF, retinoic acid, sonic hedgehog and taurine, and/or transfecting the cells with a gene for the photoreceptor cell specific transcription factor, Crx. Second, co-culture treated bone marrow-derived stem cells with embryonic retina or transplant the cells to the subretinal space in animals depleted of photoreceptor cells. Third, assess histologically the differentiation of bone marrow-derived stem cells in the retinal co-cultures or after transplantation to the retina by determining their laminar distribution in the host retina and by immunohistochemistry with antibodies specific to retinal cell types.