The development of a complex multicellular organism from a single fertilized egg requires numerous rounds of cell division. While all mitotic cells are subject to internal checkpoint controls, those in a multicellular organism are also regulated by external influences. In a developing embryo, for example, cell division must be coordinated with other developmental processes, such as cell fate specification, cell migration, and cell differentiation. The objectives of this research are to examine the temporal and spatial controls of cell division during embryonic development. These studies will be conducted in Helobdella leech embryos, which undergo invariant patterns of asynchronous and unequal cell division to produce individually identifiable cells whose cell lineages, cell cycle properties, and developmental fates are known. The first part of this research project will investigate how the timing of cell division is regulated within the different classes of leech identified cells. In particular, the patterns of expression, regulation of activity, and function of a highly conserved cell cycle control protein (Cdc25) will be examined. For example, preliminary data revealed that overexpression of Cdc25 induces premature cell divisions in the segmental founder cells. Evidence suggests that neither the absolute level of Cdc25 nor the age of the cell determines the onset of premature division, but rather premature division is triggered when these cells reach a particular location in the embryo.
Experiments are designed to identify the source of this external signal. The second part of this research will investigate how the orientation of cleavage is regulated in a specific subset of early blastomeres. Previous work has shown that, in the absence of zygotic transcription, the mitotic spindles of these cells do not assume the proper orientation for unequal cleavage and, as a consequence, they always cleave equally. In order to identify the zygotically expressed genes involved in spindle positioning, a pool of differentially expressed cDNAs has been generated using subtractive hybridization and differential screening techniques. A series of screening procedures will be used to identify putative "spindle positioning" sequences among this collection of cDNAs. Upon isolation of full-length cDNAs, those involved in spindle positioning will be identified on the basis of the ability of the corresponding RNA to restore unequal cleavages in these cells of transcriptionally inhibited embryos. These studies will contribute to the emerging appreciation of the diverse mechanisms used by cells in complex organisms to regulate the timing and symmetry of cell division.
