CHARACTERIZATION OF HOMEOBOX GENES IN THE LEECH

Many cells in advanced metazoans, particularly neurons, acquire unique, position-specific identities during development. Work with the fruit fly, Drosophila melanogaster, has led to the identification of many of the genes involved in this fundamental process. About 30 of these have been shown to have direct DNA sequence homology in a region called the homeo box that has been shown to code for a DNA-binding, conserved sequence of 60 amino acids. Cross-hybridization of genomic DNA from various other species with Drosophila probes has shown that the homeo box is conserved in evolution and has led to the isolation of homologous genes in frogs, mice and humans. Because most of the Drosophila homeo box genes affect developmental processes, in particular segmentation and segmental differentiation, it has been proposed that the homeo box could also be a marker of such genes in other species. Recent work also shows high levels of expression in the nervous system and suggests a role for these genes in the determination of neuronal fate and in differentiation. A group of animals in which a test of these ideas is particularly appropriate is the leeches. Leeches, like other annelids and arthropods, display an obvious segmental organization of their bodies. Current evolutionary theory has the arthropods and the annelids descending from a common, unsegmented ancestor, suggesting the possibility that the two groups may have cognate genes with similar or related functions. Our previous observations have demonstrated the existence of leech homeo box genes and their expression, particularly in the central nervous system, in spatiotemporal patterns like those found for homeo box genes in vertebrates and for homeotic genes in the fly. Nucleotide sequence similarities indicate that these may be leech cognates of fly homeotic genes and are, therefore, excellent candidates to be among the genes that specify segmental identity in the leech. The central aim of this project is to characterize leech genes of this class and to explore their functions by interfering with their expression. In particular, we will exploit the ability to work with identified leech neurons to determine which express these genes and to search for phenotypic differences that correlate with differential expression patterns. The techniques to be employed in this work include standard molecular cloning, in situ hybridization, construction of fusion proteins and raising antibodies, antisense RNA injection, immunohistochemistry, intracellular dye injection and electrophysiological recording, and computer-aided structural analysis. The results of this investigation should have a significant bearing on our understanding of the evolution of genes that control early development and how these genes may control neuronal phenotype.