The overall goal of this proposal continues to be to further our understanding of neuron-target cell interactions during the development of the mammalian central nervous system. The proposal exploits the power of genetics and experimental embryology to allow fresh looks at basic problems in neurobiology. The planned experiments will explore issues in two main topic areas: control of dendritic growth and regulation of nerve cell number through the process of naturally occurring cell death.Chimeras will be made with each of three neurological mutants: lurcher, staggerer, and weaver. The first two mutants have cell-autonomous defects in the Purkinje cell while weaver has a cell autonomous defect in the granule cell. One unique feature of mammalian chimeras is that the ratio of the two genotypes of cells (mutant and wild-type) can vary from mostly mutant to mostly wild-type. Thus various combinations of defective cells and healthy partners can be studied in the nervous system of a single mouse. Mathematical models that describe the cell death process will be refined and specific predictions of the models will be tested, particularly in weaver chimeras. Tritiated thymidine labeling of early vs. late born granule cells will be done in staggerer and weaver chimeras to extend studies of the role of timing of contact in the stabilization process. The numerical dependence of inferior olive neurons on the number of Purkinje cell targets will further defined in the context of the process of the polyneuronal innervation that applies to this circuit. In studies of dendritic morphology, the Golgi impregnation studies of staggerer chimeras will be concluded and compared to previous adult studies of lurcher. Lurcher chimeras will be identified at young postnatal ages through the use of RFLP analysis and analyzed to assess whether the observed dendritic alterations are due to a failure of dendritogenesis or to an atrophy of previously formed dendritic arbor. Golgi impregnations of weaver chimeras will illustrate the effects of a graded decrease in granule cell number on the form of the Purkinje cell dendritic tree. All of these studies will be aided significantly by the establishment of embryonic stem cell (ES cell) lines of the various mutant genotypes. These lines will then be used to create chimera of unambiguous genotype in greater numbers than ever before possible. Finally, the powerful technology of transgenic mouse production will continue to be used to create new markers for cells in chimeric mice. Overall, this combined genetic and morphometric approach will offer unprecedented insights into the ways in which reciprocal interactions between neuron and target cell guide the development of each. The malformations that result from the inclusion of mutant cells in the interaction will provide important hints to the pathogenesis of neurologic disease in humans as well as mice.
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