The differentiation of tissues in complex eukaryotic organisms is a central issue in developmental biology. Genetically identical cells, descendants of a single fertilized egg, somehow develop diverse forms and functions as a result of tissue-specific expression of their genomes. My laboratory makes use of a particularly simple eukaryote, Dictyostelium discoideum, to study cell interactions and cell-specific gene expression. Dictyostelium amoebae exhibit an extraordinary life cycle in which one hundred thousand independent, starved cells coalesce into mounds, and then synchronously differentiate into just two cell types, spore and stalk.
We employ a variety of approaches to questions of cell differentiation and pattern formation. Amoebae expressing foreign "reporter genes" such as GFP can be isolated and forced to develop under constrained circumstances to assess their developmental potential. The function of particular genes can be assessed by targeted gene "knock-outs" achieved by homologous DNA recombination. Some genes of particular interest to us participate in the ubiquitin-dependent pathway of regulated protein turnover; these may act by altering concentrations in the cells of the key signaling molecule, cyclic AMP. Techniques routinely used in the lab center upon gene cloning methodologies, the transformation of amoebae with foreign DNA, and the examination of expressed messenger RNA and proteins by quantitative RT-PCR, western blotting, and fluorescence. Enzymatic assays of the degradation of cyclic AMP are also performed.