Characterization of hairy/E( spl)- and eve-class genes in the posterior growth zone of the leech Helobdella robusta, a segmented lophotrochozoan

My overall research goal is to characterize hairy/ E(spl)- and eve-class genes in leech, in particular to understand how they function in the posterior growth zone of the glossiphoniid leech, Helobdella robusta. Leeches are an experimentally useful example of segmented animals that, in contrast to Drosophila, form segments sequentially rather than simultaneously along the A-P axis, and do so in an environment of mononucleate cells rather than within a syncytial blastoderm.;In Helobdella, segments arise in anterior-to-posterior progression from a posterior growth zone consisting of five bilaterally paired embryonic stem cells, called teloblasts. Each teloblast gives rise to blast cells (segmental founder cells) that contribute segmentally iterated sets of definitive progeny, in a largely cellautonomous manner. An advantage of studying the posterior growth zone in Helobdella embryos is that each teloblast is individually identifiable and its lineage is known in considerable detail.;I have identified Helobdella robusta homologs ( Hro-hes and Hro-eve) of hairy/ E(spl) and even-skipped genes, respectively, and characterized their normal expression patterns of mRNAs and proteins. Semiquantitative RT-PCR revealed that mRNA expression of Hro-hes peaks during stage 7, when all the teloblasts are making segmental founder cells; that of Hro-eve starts to increase at early stage 9 and continues to rise through late stage 10, during neurogenesis.;In situ hybridization (and immunostaining) revealed that both genes are expressed throughout early development until stage 8, in all types of cells, including proteloblasts, macromeres and micromeres, as well as the teloblasts and the primary blast cells. HRO-HES is localized in nuclei of cells expressing HRO-HES during interphase but declines during mitosis. Transcripts levels of Hro-hes (and Hro-eve) peak during mitosis, cycling in anti-phase to HRO-HES protein, and these mitotic transcripts are associated with the chromatin. Thus, the nuclear localization of Hro-hes transcripts and protein is reciprocal during cell cycle. Down-regulation of Hro-hes expression by injecting antisense oligonucletide, diverts cell OP to a purely teloblastic state by blocking its equal cell division. Together, these results suggest that Hro-hes and Hro-eve are important in regulating stem cell cycle and fate decisions in leech.;Late expression of Hro-eve mRNA at stages 9--10 occurs dynamically in segmentally iterated sets of ganglionic neurons in the ventral nerve cord. Lineage analysis revealed that neurons expressing Hro-eve arise from the N-derived cells.