Richard L. Sidman, MD - US grants

This long-term grant has contributed many of the primary analyses of disease phenotypes in mice with inherited neurological diseases, and thus has provided model systems for the analysis of developmental cell interactions in the complex nervous system of mammals. It has also provided core material for experimental study of disease mechanisms relevant to our understanding of human degenerative diseases of the nervous system. The mouse, for technical reasons, is by far the most relevant animal species for the many types of genetic studies that are now at the center of biomedical science. A primary research aim for the forthcoming five-year cycle involves the continuation of our studies on cell interactions in development. We will turn to the early embryo to test the hypothesis that the first incoming axonal systems influence the deployment of the early-forming neuronal populations that are migrating into the developing cerebellar cortex. We propose that also in the first weeks after birth, when mice arc still very immature, a similar set of interactions in time and space between incoming axons and at least two major neuronal populations mediate the acquisition of adult cerebellar organization. The data relevant to test these ideas will come from new methods for examining quantitatively and efficiently the numbers, and the three dimensional positions and configurations, of cellular elements in whole mounts and thick slices of tissue examined by confocal microscopy. We will use as our experimental material a large series of available mouse mutations that perturb the development or viability of neurons and allow us to explore developmental mechanisms in the relatively inaccessible immature mammalian cerebellum. Further, we will seek a specific mechanism in the nr mutant, where we hypothesize that cerebellar granule cells may mediate the death of related Purkinje neurons through a toxic action of excitatory neurotransmitter molecules. We will also seek new insights into late-life neurodegenerative diseases by examining cell relationships in several mutants where Purkinje neurons die relatively early in life, and then presynaptic granule cell neurons die secondarily at much later ages. A final aim is to continue the description of new inherited mouse disorders, and to develop, characterize, and distribute the specialized genetic stocks necessary to make full use of the potential of the science of genetics for increasing our understanding of the formation, organization, and diseases of the nervous system.

The long-term aims of this project are 1) to establish the genetic control points governing assembly of the retina, a complex but relatively accessible part of the central nervous system, and 2) to provide new ideas that may lead to the prevention and treatment of inherited human diseases of the retinitis pigmentosa group. The essential feature of these diseases is a progressive degeneration of retinal photoreceptor cells. The project concentrates on describing and analyzing several new inherited diseases causing a similar loss of retinal photoreceptor cells in the mouse, the most favorable mammalian species, along with man, for analyzing genetic disorders. In the forthcoming three years, we propose to present the initial description of diseases caused by three independent mouse genes, named wabbler lethal-2J (wl2J), vitiligo (vit), and hugger (hug). The wabbler lethal disorder may be present in two forms caused by independent mutations, wl and wl2J, both at the wabbler lethal locus, but this will be clear only when the wl mice are bred so as to eliminate a complicating disease that prevents expression of the putative wl retinal disorder. The plan of study involves development of comparable, uniform genetic stocks so that the diseases can be compared quantitatively and reliably with one another and with previously described mouse diseases that are somewhat like human retinitis pigmentosa, but not identical. The vit and hug genes will be mapped, wl having already been mapped to a position about one-third of the distance from the centromere to the far end of chromosome 14. The timing of the photoreceptor cell loss will be measured from microscope specimens, and the cause of the cell loss will be sought by immunocytochemical and tissue grafting methods. The most promising clue at present is that all three diseases involve defects in length and integrity of rod outer segments, the part of the photoreceptor cell that contains the light-absorbing pigment. Mechanisms controlling rod outer segment growth and structure will be sought, in the hope of intervening to slow the pace of the disease. Mice will be made available to other investigators upon request.

The aims of this grant are (1 to place mutations affecting the retina in mice onto a few selected genetic backgrounds by standard breeding schedules, so that the gene actions can be more accurately compared and contrasted with one another, 2) to distribute affected and control mice or tissues or chemical extracts to qualified research scientists at no charge to them except for shipping costs, so that they can carry on their research without committing the time or bearing the expense of generating their own mice, and 3) to mutagenize male mice and mate them with females carrying known retinal mutations, so as to select for new mutant alleles that will illuminate how particular genes control retinal function and cause disease. Progeny of mutagenized mice will be screened for retinal disease by nondestructive indirect ophthalmoscopy, a rapid and effective method that markedly reduces the need to screen laboriously by expensive histological methods.These mouse models have been adding significantly to our understanding of inherited diseases of the retinitis pigmentosa category in humans, and are likely to contribute even more crucially in the years immediately ahead. The most widely used mutations at present are named retinal degeneration (rd), retinal degeneration slow (rds), Purkinje cell degeneration (pcd), and nervous (nr). We maintain each of these on several genetic backgrounds, including three independently-occurring mutant alleles at the pcd locus. We have developed, or are developing, most of these strains, and are the unique holder of several of them. Additionally, we will supply mice with new retinal disorders named hugger (hug), vitiligo (vit), and wabbler lethal-2J (wl2J); these affect not only retinal photoreceptor cells, the hallmark of the retinitis pigmentosa class of diseases, but also affect other components of the retina, and thus open the prospect of deepening our understanding of genetic control and functions of cells in retinal pigment epithelium and inner nuclear and ganglion cell layers. Further available mutants serve as models of congenital stationary night blindness. Still others cause hypopigmentation and developmental abnormalities in the trajectories of ganglion cell axons in the optic nerve. We are prepared also to advise other investigators, as we have done during the first funding cycle of this grant, concerning what mutants, genetic backgrounds, controls, ages, and numbers of mice might help to solve their research questions. Another service function will be to take on the propagation and dissemination of mutant stocks, including transgenic mice with retinal abnormalities, that are developed by other investigators who may not wish to remain solely responsible for holding and distributing such research material.

The aims of this grant are (1 to place mutations affecting the retina in mice onto a few selected genetic backgrounds by standard breeding schedules, so that the gene actions can be more accurately compared and contrasted with one another, 2) to distribute affected and control mice or tissues or chemical extracts to qualified research scientists at no charge to them except for shipping costs, so that they can carry on their research without committing the time or bearing the expense of generating their own mice, and 3) to mutagenize male mice and mate them with females carrying known retinal mutations, so as to select for new mutant alleles that will illuminate how particular genes control retinal function and cause disease. Progeny of mutagenized mice will be screened for retinal disease by nondestructive indirect ophthalmoscopy, a rapid and effective method that markedly reduces the need to screen laboriously by expensive histological methods.These mouse models have been adding significantly to our understanding of inherited diseases of the retinitis pigmentosa category in humans, and are likely to contribute even more crucially in the years immediately ahead. The most widely used mutations at present are named retinal degeneration (rd), retinal degeneration slow (rds), Purkinje cell degeneration (pcd), and nervous (nr). We maintain each of these on several genetic backgrounds, including three independently-occurring mutant alleles at the pcd locus. We have developed, or are developing, most of these strains, and are the unique holder of several of them. Additionally, we will supply mice with new retinal disorders named hugger (hug), vitiligo (vit), and wabbler lethal-2J (wl2J); these affect not only retinal photoreceptor cells, the hallmark of the retinitis pigmentosa class of diseases, but also affect other components of the retina, and thus open the prospect of deepening our understanding of genetic control and functions of cells in retinal pigment epithelium and inner nuclear and ganglion cell layers. Further available mutants serve as models of congenital stationary night blindness. Still others cause hypopigmentation and developmental abnormalities in the trajectories of ganglion cell axons in the optic nerve. We are prepared also to advise other investigators, as we have done during the first funding cycle of this grant, concerning what mutants, genetic backgrounds, controls, ages, and numbers of mice might help to solve their research questions. Another service function will be to take on the propagation and dissemination of mutant stocks, including transgenic mice with retinal abnormalities, that are developed by other investigators who may not wish to remain solely responsible for holding and distributing such research material.

This long-term grant has contributed many of the primary analyses of disease phenotypes in mice with inherited neurological diseases, and thus has provided model systems for the analysis of developmental cell interactions in the complex nervous system of mammals. It has also provided core material for experimental study of disease mechanisms relevant to our understanding of human degenerative diseases of the nervous system. The mouse, for technical reasons, is by far the most relevant animal species for the many types of genetic studies that are now at the center of biomedical science. A primary research aim for the forthcoming five-year cycle involves the continuation of our studies on cell interactions in development. We will turn to the early embryo to test the hypothesis that the first incoming axonal systems influence the deployment of the early-forming neuronal populations that are migrating into the developing cerebellar cortex. We propose that also in the first weeks after birth, when mice arc still very immature, a similar set of interactions in time and space between incoming axons and at least two major neuronal populations mediate the acquisition of adult cerebellar organization. The data relevant to test these ideas will come from new methods for examining quantitatively and efficiently the numbers, and the three dimensional positions and configurations, of cellular elements in whole mounts and thick slices of tissue examined by confocal microscopy. We will use as our experimental material a large series of available mouse mutations that perturb the development or viability of neurons and allow us to explore developmental mechanisms in the relatively inaccessible immature mammalian cerebellum. Further, we will seek a specific mechanism in the nr mutant, where we hypothesize that cerebellar granule cells may mediate the death of related Purkinje neurons through a toxic action of excitatory neurotransmitter molecules. We will also seek new insights into late-life neurodegenerative diseases by examining cell relationships in several mutants where Purkinje neurons die relatively early in life, and then presynaptic granule cell neurons die secondarily at much later ages. A final aim is to continue the description of new inherited mouse disorders, and to develop, characterize, and distribute the specialized genetic stocks necessary to make full use of the potential of the science of genetics for increasing our understanding of the formation, organization, and diseases of the nervous system.

The long-term aims of this project are 1) to establish the genetic control points governing assembly of the retina, a complex but relatively accessible part of the central nervous system, and 2) to provide new ideas that may lead to the prevention and treatment of inherited human diseases of the retinitis pigmentosa group. The essential feature of these diseases is a progressive degeneration of retinal photoreceptor cells. The project concentrates on describing and analyzing several new inherited diseases causing a similar loss of retinal photoreceptor cells in the mouse, the most favorable mammalian species, along with man, for analyzing genetic disorders. In the forthcoming three years, we propose to present the initial description of diseases caused by three independent mouse genes, named wabbler lethal-2J (wl2J), vitiligo (vit), and hugger (hug). The wabbler lethal disorder may be present in two forms caused by independent mutations, wl and wl2J, both at the wabbler lethal locus, but this will be clear only when the wl mice are bred so as to eliminate a complicating disease that prevents expression of the putative wl retinal disorder. The plan of study involves development of comparable, uniform genetic stocks so that the diseases can be compared quantitatively and reliably with one another and with previously described mouse diseases that are somewhat like human retinitis pigmentosa, but not identical. The vit and hug genes will be mapped, wl having already been mapped to a position about one-third of the distance from the centromere to the far end of chromosome 14. The timing of the photoreceptor cell loss will be measured from microscope specimens, and the cause of the cell loss will be sought by immunocytochemical and tissue grafting methods. The most promising clue at present is that all three diseases involve defects in length and integrity of rod outer segments, the part of the photoreceptor cell that contains the light-absorbing pigment. Mechanisms controlling rod outer segment growth and structure will be sought, in the hope of intervening to slow the pace of the disease. Mice will be made available to other investigators upon request.

The goal of this project is to produce a high resoluation electronic atlas of the brain in three dimensions (3D). A number of technical problems are addressed, including how to minimize distortions during the processing of the brain through steps of hardening (fixation), embedding in a rigid support medium, sectioning and staining. Other problems relate to the massive computer database and memory requirements for the large number of images at the closely spaced intervals through the brain necessary for resolution of 1 um or less in all three planes of space (1 um isomorphic voxels). To solve these problems, we propose to make an atlas of the small brain of the mouse, as a prelude to tackling the human brain. We will begin at a voxel resolution of 10 um, and procede to 3 um and 1 um (greater than 550 GB of image data). Novel technical methods for whole brain embedding and sectionaing in epoxy resin and for confocal microscopy of stable 3D data space, and for partially automated volumetric segmentation of brains packed with hundreds of 3D structures, many with comples shapes. Images will be warped to a high resolution MRI standard of the intact fixed head (100 um digital "slices" obtained with a 9.4 Tesla magnet). The uniquely high level of histological resolution in 3D will allow us to develop a new "white matter anatomy" by tracing the exact trajectories of myelinated fiber groups that intermingle so intimately in the white matter that they have been impossible to sort out by previously available characterization of brain structures. Chemical, genetic, and pathological data will be superimposable digitally onto the basic cell and myelin atlas templates. For ease of data storage and transmission, the atlas images will be compressed with wavelet compression algorithms, with an added substraction method that will allow the origical focus on the elucidation of the genetic gases for individual differences in brain organization. Remote users with their own research, educational, and public welfare needs, will be able to obtain the entire database, including capabilities for rotations and digital "resectioning" in any desired orientation, on new high capacity DVD disks, or will gain access to specific 2D and 3D images via a dedicated WWW page outfitted with a systematic and friendly query interface and VRML-based browser. Users needing to download atlas images will access the WWW pageover new high speed Asynchronous Transfer mode transmission lines.