Research at Loomis Lab
CELL-CELL COMMUNICATION DURING DEVELOPMENT
Temporal and spatial patterns of gene expression underlie development in all organisms but are more easily analyzed in those systems where high resolution genetic techniques can be applied. We focus on cell differentiation in Dictyostelium discoideum where a complete physical map is available and we expect to have the complete sequence of the 34 Mb genome in the next few years. Annotation efforts are now underway in the lab to generate a Preliminary Directory of Dictyostelium genes and to define the proteome.
Aggregates of 105 cells are formed within the first 12 hours following the initiation of development and fruiting bodies complete with spores and a cellular stalk are formed in the next 12 hours. Gene expression analyses using microarrayed DNA from hundreds of genes has shown that clustersof genes are expressed at specific stages during the 24 hour developmental cycle. Cell type specific genes were recognized by probing with polyA+ mRNA isolated from either prespore or prestalk cells. These genes can be used to define the cell types and follow their divergence and regulation. Microarray expression analyses on strains carrying mutations in genes encoding components of the signal transduction pathways will define the genetic networks that underlie development in this system.
Using random plasmid insertion mutagenesis (REMI) we have discovered over 150 developmental genes many of which function during development of flies, worms, and mammals while others are novel. A combination of biochemical and genetic techniques are used to understand the mechanisms responsible for orderly progression through the stages as well as proper proportioning of the cell types. Saturation screens for suppressor mutations can uncover surprising connections between genes in different parts of the networks.
We are focusing on the components responsible for changing solitary amoebae into chemotactically interacting cells that adher to each other prior to forming aggregates. These include seven-transmembrane G-protein coupled cAMP receptors, adenylyl cyclases, cAMP phosphodiesterases, MAP kinases and the cAMP dependent protein kinase, PKA. Together they form a spontaneously oscillating circuit that regulates signal relay as well as cell motility.
We are also analyzing the signal transduction pathway that integrates prespore and prestalk differentiation. During culmination a peptide signal, SDF-2 , is released and stimulates terminal differentiation of spores. It is bound by the surface receptor, DhkA, a histidine kinase that leads indirectly to the inhibition of the cytoplamic cAMP phosphodiesterase, RegA. As cAMP levels go up, the cAMP dependent protein kinase PKA is activated and triggers fusion of prespore vesicles with the plasma membrane releasing spore coat proteins. While the details may be specific to Dictyostelium , we feel that the types of interactions that coordinate differentiation in both time and space may be common to many multicellular organisms.
Rappel, W-J., Nicol, A., Sarkissian, A., Levine, H. and Loomis, W.F. (1999) Self-organized vortex state in two-dimensional Dictyostelium dynamics. Phys. Rev. Letters 83, 1247-1250.
Nicol, A., Rappel, W-J., Levine H. and Loomis, W.F. (1999) Cell-sorting in aggregates of Dictyostelium discoideum. J. Cell Sci. 112, 3923-3929.
Söderbom, F., Anjard, C., Iranfar, N., Fuller, D. and Loomis, W.F. (1999) An adenylyl cyclase that functions during late development of Dictyostelium Development 126, 5463-5471.
Loomis, W.F. and Insall, R. H. (1999) A cell for all reasons. Nature 401, 440-441.
Blanton, R.L., Fuller, D., Iranfar, N., Grimson, M.J., and Loomis, W.F. (2000) The cellulose synthase gene of Dictyostelium. Proc. Natl Acad. Sci. 97, 2391-2396.
Wessels, D., Zhang, H. Reynolds, J., Daniels, K., Heid, P., Liu, S., Kuspa, A., Shaulsky, G., Loomis, W.F., Soll, D.R. (2000) The internal phosphodiesterase RegA is essential for suppression of lateral pseudopods during chemotaxis of Dictyostelium Mol. Biol. Cell 11, 2803-2820.
Wang, J., Hou, L., Awrey, D., Loomis, W.F., Firtel, R.A., and Siu, C-H. (2000)
Membrane glycoprotein gp150 is encoded by the lagC Gene and mediates cell-cell adhesion via heterophilic binding during Dictyostelium development Devel. Biol. 227: 734-745
Sasik, R., Hwa, T. Iranfar, N., and Loomis, W.F. (2001) Percolation clustering: a novel approach to the clustering of gene expression patterns in Dictyostelium development. Pacific Symp. Biocomputing ed.R. B. Altman, A. K. Dunker, L. Hunter, K. Lauderdale, and T. E. Klein, World Scientific, Singapore (pp. 335-347).
Anjard, C., Soderbom, F. and Loomis, W. F. (2001) Requirements for the adenylyl cyclases in development of Dictyostelium. (submitted).
Zhang, P., McGlynn, A., Loomis, W.F., Blanton, R. L., and West, C.M. (2001) Spore coat formation and timely sporulation depend on cellulose in Dictyostelium. Differentiation (in press).
Anjard, C. and Loomis, W. F. (2001) The histidine kinases of Dictyostelium. in "Histidine Kinases in Signal Transduction" edited by R. Dutta and M. Inouye. Acad. Press, San Diego (in press).