John A. Freeman - US grants

The principal objective of the proposed research is to identify and characterize some critical cellular mechanisms controlling the growth of optic nerve fibers and the formation of retinotectal synapses, using a combination of morphological, physiological, and biochemical techniques. The retinotectal system provides a very favorable model for studying the dynamics of neurite growth and synaptic development at the cellular and molecular levels, and we plan a set of comparative studies in vivo and in vitro in teleosts (goldfish) and mammals (rats). There are three major goals. The first is to investigate the dynamics of neurite growth in vitro. We have recently discovered that retinal neurite growth cones generate steady currents across their tips, and grow in the direction of an applied field. We plan to examine the effects of local electrical events on cell-substrate adhesion, growth, and the movement and assembly of membrane macromolecules, using internal reflectance and video-enhanced microscopy. Electrogenesis of growing neurites will be monitored with conventional microelectrode recording methods and by the use of a voltage-- sensitive dye. The second goal is to study the development of retinotectal synapses using quantitative EM morphometric and three-dimensional reconstruction methods in order to correlate structural changes and changes in the distribution of membrane macromolecules seen using freeze-fracture methods, with the development of synaptic transmission. The third goal is to establish optimal homogenous culture condtions, by isolating different cell types, and by establishing a continuous cell line of retinal ganglion cells. These studies will involve the development of specific antibodies to different cell types, the production of which would be of great use in the future as tools for cell identification and separation, as molecular probes for membrane macromolecules, and in providing an animal model for autoimmune retinopathies. These studies embrace an area of research (retinal regeneration) targeted by a recent report of the National Advisory Eye Council as being of special interest.

Recent work has led to the discovery of a class of growth-associated proteins, called GAPs, whose expression is selectively enhanced in regenerating and developing neurons of some species. The purpose of this investigation is to study the cellular role of these growth-associated proteins, and in particular to determine whether tje abo;otu pf a meirpm tp regenerate depends on its ability to induce their synthesis. There are five related objectives. The first objective is to determine, by means of a limited phylogenetic survey, whether these proteins appear in CNS neurons lacking the capability to regenerate, or whether they occur only in growing or regenerating neurons. Two very sensitive and powerful techniques will be employed to detect and quantitate these proteins: computer-analyzed 2-dimensional gel electrophoresis, and immunological techniques. The second objective is to determine the cellular localization of GAPs, using EM immunocytochemical localization methods. The third objective is to characterize the major distinguishing characteristics of GAPs, and the fourth is to investigate the cellular functions of GAPs, by correlating the kinetics of GAP expression with different phases of axonal growth in developing and regenerating systems. The last objective is to determine how the expression of GAPs is regulated. We plan to study the effects of several likely regulatory molecules on GAP synthesis, including nerve growth factor (NGF) and cyclic AMP. The site of regulation (transcriptional, translational, of post- translational) will be determined with inhibitors of RNA and protein synthesis. Finally, specific changes in GAP messenger RNA during regeneration will be observed using a cell-free synthesis system. The significance of these studies lies in their promise to reveal some molecular mechanisms which control axonal growth. These are of paramount importance in understanding the development of the nervous system, and its response to injury.

Growth of organ primordia during post-gastrula morphogenesis, along with cell shape change and cell differentiation, leads to the form and shape of the developing tissue. Although cell replication occurs throughout the organ forming period in most tissues, its role in generating the force for forming the three dimensional shape of the tissue such as the evagination of a tube from an epithelial sheet, is not well understood. Factors that have hindered comprehension of the potential of cell division for shape generation include the previous inability to experimentally separate mitotic pressure from other shape generating processes and the lack of a system in which the process can be followed in situ. In the proposed study the role of cell replication in generating both the number and pattern of cells in the epithelium of a segment, as a stimulant for cell differentiation of specific cell types within the epithelium, and as a motive force in the shape change of the epithelium during limb bud formation will be investigated using larvae of the brine shrimp, Artemia. The simplicity of the tissue organization of these larvae makes this organism ideally suited for this study. The cells involved in the limb bud and segment forming regions are few in number, clearly defined, not surrounded by other cells, and have well timed growth periods. Analysis of the experiments will be carried out using both video-based image analysis and immunocytochemistry. The following hypotheses will be tested: 1) Cell replication occurs in a spatio-temporally directed manner that results in a set pattern of cells and regions of unequal cell density. 2) Attainment of this pattern leads to the differentiation of arthrodial membrane and tendonal cells in which microtubules are involved in cell shape change. 3) Deformation of the epithelium and evagination of the thoracopod limb bud result form unequal cell density, the presence of differentiated arthrodial membrane cells, and region-specific apolysis. 4) Maintenance of the form and shape of the limb bud involves continued patterned cell replication, new cuticle formation, and tendonal cell differentiation. The role of microtubules and microfilaments in the evagination process will also be determined. Some sections of the study will provide preliminary evidence necessary for future studies on regulation of cell replication during development of segmental structures.