CHARACTERIZATION OF HOMEO BOX HOMOLOGIES IN THE LEECH

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It has been recently demonstrated that there is direct DNA sequence homology between several Drosophila homeotic genes. This region has been called the homeo box and has been shown to code for a conserved, about 60 amino acid residue sequence. Cross-hybridization of genomic DNA from various other species with probes containing the Drosophila homeo box has suggested that regions homologous to the homeo box are evolutionarily conserved. Cross-hybridization has also been useful in the isolation of some of the genes that contain this region in frogs, mice and humans. Because the Drosophila genes that contain the homeo box affect developmental processes, in particular segmentation and segmental differentiation, it has been proposed that the homeo box could also be a marker for such genes in other species. A group of animals in which a test of this idea would be particularly appropriate are leeches. Leeches, like all other annelids and like the insects, display an obvious segmental organization of their bodies. Current evolutionary theory has the insects descending from an annelid ancestor, suggesting the possibility that the insect homeotic genes have evolved from corresponding annelid genes. Furthermore, our preliminary observations indicate that leeches also have genes that contain regions which cross-hybridize with the fly homeo box. The aim of the project proposed here is to identify and isolate such genes in a few species of leeches, and to determine whether their spatial and temporal patterns of expression are similar to those of the Drosophila genes that contain the homeo box. We will pursue this goal by first determining the approximate number of homeo box cross-hybridizing regions in the leech by Southern blot analysis. We will then determine the degree of homology with the homeo box by sequencing the regions of leech DNA of interest and will characterize their transcripts by Northern analysis. Finally, we will do in situ hybridization on serial tissue sections of leech embryos to assess the spatial and temporal patterns of expression of the identified leech genes. In particular, we will study the expression of these genes at the level of individual, identified neurons in the leech CNS, to determine whether differential gene expression correlates with the segmental differentiation of neuronal phenotypes that we have characterized. The results of this investigation should have a significant bearing on our understanding of the evolution of genes that control early development and of the role of such genes in the genesis of phenotypic heterogeneity in the CNS.