Cell motion plays a critical role during embryogenesis in virtually all animals. Until now, studies of cell movement have been limited to a two-dimensional analysis, even though morphogenesis is almost invariably three dimensional. To visualize the 3-D motion of cells in vivo, I have assembled a sophisticated computer-assisted microscope. This microscope is capable of obtaining a 3-D series of optical sections at very low light levels, thus permitting long-term, three-dimensional observation of a living specimen. The techniques I have developed now permit visualization of the 3-D motion of any cell that can be tagged with a fluorescent marker. I am applying these techniques to the study of cell motion during morphogenesis in the cellular slime mold Dictyostelium discoideum. In D. discoideum, amebae aggregate to form a multicellular mass that undergoes dramatic shape changes in subsequent development. These global shape transformations depend critically on cell movement because they occur in the absence of cell division or marked cell-shape changes. Morphogenesis eventually concludes with overt cell differentiation into one of two different cell types: stalk cell or spore cell. Prestalk cells are found in the anterior of the cell mass while prespore cells are found in the posterior. This partitioning of the two cell types is likely due, at least in part, to the sorting of cells pre-destined to pursue one or the other differentiation pathway. How this segregation of the two cell types occurs is unknown, but two different models have been proposed. In one model, prestalk cells sort to the anterior by chemotaxis, while in the other model prestalk and prespore cells separate into two phases based on differential adhesive preferences. Each of these models predicts distinct motile behaviors for prestalk and prespore cells. Indeed, in preliminary experiments, I have already obtained the first in vivo evidence for at least two distinct types of 3-D cell motion during morphogenesis in D. discoideum. I now propose to extend these experiments by examining specifically prestalk and prespore cell motion within the cell mass to distinguish between the two models for cell sorting. In addition, I wish to examine cell motion in various mutants or transformants with defects in chemotaxis or intercellular adhesion. Inspection of 3-D cell motion in these mutants or transformants will provide evidence for or against the chemotactic or differential adhesion models. The significance of these studies to visualize cell motion in 3-D is threefold. First, in D. discoideum, the analysis will provide an answer to longstanding questions about how cell sorting occurs during morphogenesis. Second, in D. discoideum the technique will also provide an assay to unravel the molecular mechanisms that govern cell movement, sorting and assembly during morphogenesis. Third, the method is likely to find broad application to the study of cell movement in a variety of organisms.
