The long-term goal of this research is to elucidate the role of the Antennapedia class of homeobox genes in the segmental diversification of identified neurons. These genes have been shown to govern the establishment of segment identity in a wide variety of organisms. There has, however, been little effort to investigate their function at the level of single identified cells, in particular to ascertain whether the transcription factors expressed by these genes are necessary and sufficient to switch neurons between alternative fates appropriate to different segments. This problem will be addressed in the leech embryo because (unlike other experimental organisms) it is straightforward to examine the expression of these genes in identified, segmentally homologous neurons at the time when those neurons are first beginning to manifest segment-specific patterns of differentiation. Gene expression will be monitored by staining the central nervous system of dissected leech embryos with antisera specific to the protein products produced by three different homeobox genes: Lox2, Lox5 and Lox6. Homologous neurons situated in different body segments will be examined to ascertain whether or not there is a direct correlation between segmental differences in neurotransmitter phenotype or programmed cell death and segmental differences in homeobox gene expression. Gene expression will also be examined in a set of neuronal progenitor cells which are only found in certain body segments, and compared with the expression pattern of developmentally homologous cells (which do not generate neuronal progeny) situated in other segments. To investigate gene function, the normal pattern of Lox gene expression will be modified in developing leech embryos. The Lox genes will be transiently expressed in ectopic segments by the injection of mRNA or transgenes into identified segmental precursor cells. In segments where a gene is normally expressed, protein accumulation will be reduced by injection of antisense oligonucleotides targeted to prevent the translation of that mRNA. Changes in the segment identity of the transfected cell lineage would suggest that the gene in question is responsible for segmental diversification. Similar techniques will also be applied to adult leech neurons which have been isolated from the nervous system and raised in culture. This latter experiment will test whether persistent expression of the Lox genes is necessary for the maintenance of segment identity in a fully differentiated neuron. If it is found that these particular homeodomain transcription factors do in fact govern the segment identity of single neurons in a manner which directly reflects the segmental distribution of the gene's expression, it would represent a major advance in the understanding of how neuronal identity is determined by regulatory gene expression, and provide a model for addressing the role of gene regulation in neurodegenerative disorders that involve the misspecification of cellular identity.
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