The proposed research will investigate factors that may be important in determining the lineages of two precociously developing neurons in the frog embryo, Rohon-Beard (RB) neurons and primary motoneurons (PMN). Quantitative fate maps for these neurons, derived from intracellular injections of the lineage marker, horseradish peroxidase, have recently been constructed for the 16- and 32-cell stage blastulae. The proposed experiments will perturb the normal blastomere arrangement in a variety of ways in order to determine, by comparison to the normal quantitative fate map, whether the phenotype and number of these two specific neurons are determined to arise from particular blastomeres, or to arise from any blastomere located in the appropriate position. These experiments are important in the study of the effect of genomic and/or cytoplasmic influences on the commitment of embryonic cells to the nervous system. Blastomeres that give rise to a known number of RB and PMN will be intracellularly injected with horseradish peroxidase. Neighboring blastomeres, whose final neuronal lineages are known, will be excised (one per experiment). The RB and PMN descendants of the injected blastomere will be tabulated to determine whether the extirpation of its neighbor caused a change in its own lineage pattern. In addition, single labeled blastomeres will be transplanted to ectopic positions in order to determine whether they will express their original phenotypes, or will express phenotypes characteristic of their new location. Finally, transplantations of nuclear, cytoplasmic, and plasma membrane constitutents from blastomeres expressing one neuronal phenotype into blastomeres expressing a different neuronal phenotype will be performed to determine in which subcellular compartment the instructions for the specific neuronal phenotype might reside. The results from these experiments will lead to the analysis of neuronal lineage determination at a molecular biological level. In this manner, we will be able to analyze what developmental decisions lead to the formation of the vertebrate nervous system.
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