Research Summary Cell Signaling, Pattern Formation, Growth Control, Cell Movement Developmental Biology The primary focus of our research is the regulation of patterning and growth during animal development, with a particular emphasis on fringe-dependent cell signaling. We are also interested in directed cell rearrangements that effect morphogenesis. Our work takes advantage of the powerful genetic, molecular and cellular techniques available in Drosophila, which facilitate both gene discovery and the analysis of gene function. Cell Rearrangement Oriented cell rearrangements elongating the primary body axis occur during the development of many animals, but the mechanism and regulation of these rearrangements is not understood. In Drosophila, a large portion of the embryo, the germband, simultaneously elongates and narrows during early embryogenesis, undergoing a 2.7 fold extension. Investigation of living embryos with time-lapse video microscopy, under conditions where individual cells could be followed, demonstrated that this extension is accompanied by directed cell rearrangements. Analysis of Drosophila patterning mutants further showed that cell rearrangement during germband extension is a regionally autonomous process that is dependent upon pair-rule segmentation genes. This led to a model in which cell rearrangement results from the establishment of adhesive differences between stripes of cells by these pair-rule genes. The aim of this project is to use a combination of genetic, molecular, and cell biological studies to elucidate the basis for these directed cell rearrangements. Cellular Interactions Directing Wing Patterning and Growth Studies of both insect and vertebrate limbs have revealed a relationship between patterning and the regulation of cell proliferation. The Drosophila wing primordia (imaginal disc) is subdivided into both anterior-posterior and dorsal-ventral compartments. Interactions between cells in different compartments are then necessary for the further growth and patterning of the wing. The fringe gene is expressed in dorsal wing cells and encodes a novel secreted protein. In collaboration with Tom Vogt at Princeton, fringe-related genes have also now been identified both in other insects and in vertebrates, suggesting that fringe actually defines a new family of cell-signaling molecules. Genetic and molecular studies of Drosophila fringe indicate that it mediates interactions between dorsal and ventral wing cells. These interactions induce both cell proliferation and the specification of specialized cells at the edge of the wing, the wing margin. Importantly, it is not the expression per se of fringe which induces wing margin formation and wing growth, but the juxtaposition of cells that express and cells that lack fringe. Cells which themselves express fringe appear unable to respond to fringe, consequently restricting fringe signaling to the boundaries of fringe expression. This project is now directed towards in-depth characterization of the fringe-signaling process. Biochemical Characterization of fringe fringe defines a new protein family, so it is not possible to make inferences about its biochemical activity by comparison to other known proteins. However, the fringe open reading frame includes a predicted signal peptide at its amino terminus and lacks predicted transmembrane domains, suggesting that it is secreted. By labeling the fringe protein with a myc epitope-tag, we have been able to confirm that fringe can be efficiently secreted when expressed in a Drosophila cell line. Another intriguing feature of the fringe amino acid sequence is the presence of potential recognition sites for dibasic processing enzymes. A number of secreted signaling proteins are first synthesized as inactive precursors and then processed by proteolytic cleavage at dibasic sites. We have been particularly intrigued by the possibility that the existence of distinct forms of fringe might account for the boundary-specific nature of its activity. One of these sites is conserved among vertebrate fringe genes. Preliminary analysis of fringe expressed in Drosophila cell lines has revealed shifts in its apparent molecular weight that are consistent with proteolytic processing at the conserved dibasic processing site. Intersection of fringe and Notch Signaling The Notch gene encodes a receptor protein that mediates a number of key cell fate decisions during both vertebrate and invertebrate development. Aberrant Notch signaling has also been linked to leukemia in humans, and to breast cancer in mice. Notch, together with the Notch ligands Serrate and Delta, also plays an essential role in dorsal-ventral cell interactions during wing development, raising the possibility that fringe may intersect with the Notch signaling pathway. In fact, we have observed that fringe affects the Notch ligand Serrate in two distinct ways: in collaboration with Sean Carroll (University of Wisconsin), we established that fringe expression boundaries induce the expression of Serrate, and we have recently demonstrated that fringe inhibits cell's ability to respond to Serrate. The observation that dorsal (fringe-expressing) cells respond to Delta but not to Serrate, while ventral (fringe nonexpressing) cells respond well to Serrate but poorly to Delta, further suggests that fringe may differentially modulate the ability of these two ligands to activate the Notch receptor. The basis for these effects is being investigated by combined biochemical, cell culture, and in vivo analysis. Analysis of fringe Activity During Eye and Leg Development Our initial understanding of fringe function derived from studies of its action during wing development, however, fringe also plays essential roles in the development of many other tissues. In addition to contributing to our understanding of Drosophila development, comparative studies of fringe function in different tissues are being pursued to determine whether the understanding we have developed of fringe activity in the wing actually reflects general principles of fringe-dependent cell signaling, as opposed to peculiarities of wing development. We aim to determine how patterns of fringe expression relate to fringe activity (e.g., is fringe always boundary-specific?), and whether fringe-dependent cell signaling is always linked to Notch activity. We have chosen to focus on the eye and the leg as two model systems for comparative studies of fringe-dependent cell signaling. These tissues offer four principle advantages for the elucidation of fringe function: (1) they exhibit a variety of different fringe expression patterns, (2) their development has been well studied, (3) multiple molecular markers exist to identify different cell fates, and (4) requirements for the activity of Notch and its ligands have been defined. Our results thus far support the proposition that fringe functions in other tissues in the same way as it does in the wing. In the eye, fringe is expressed specifically in ventral cells during early eye development, and both loss of fringe activity and uniform fringe expression lead to loss of eye tissue. Thus, as in the wing, fringe may be acting boundary-specifically to mediate interactions between dorsal and ventral cells, leading to growth of the eye primordia. In the leg, fringe appears to be expressed within cells adjacent to leg segment boundaries, and both loss of fringe activity and ectopic expression of fringe lead to fusions between leg segments. Notch, Serrate, and Delta are also required for eye and leg development, and mutation of these genes can yield similar phenotypes to those caused by fringe mutations. Publications Rauskolb C., Correia T., and Irvine K.D. (1999) Fringe-dependent separation of dorsal and ventral cells in the Drosophila wing. Nature. 401:476-80. Irvine K.D. (1999) Fringe, Notch, and making developmental boundaries. Curr Opin Genet Dev. 9:434-41. Rauskolb C, and Irvine K.D. (1999) Notch-mediated segmentation and growth control of the Drosophila leg. Dev Biol. 210:339-50. Panin V.M and Irvine K.D. (1998) Modulators of Notch signaling. Semin Cell Dev Biol. 9:609-17. Simmonds A.J., Liu X., Soanes K.H., Krause H.M., Irvine K.D., and Bell J.B.. (1998) Molecular interactions between Vestigial and Scalloped promote wing formation in Drosophila. Genes Dev. 12(24):3815-20. Papayannopoulos V., Tomlinson A., Panin V.M., Rauskolb C., and Irvine K.D. (1998) Dorsal-ventral signaling in the Drosophila eye. Science 281:2031-4. Irvine K.D. and Vogt T.F. (1997). Dorsal-ventral signaling in limb development. Curr Opin Cell Biol. 9(6): 867-876 Panin, V.M., Papayannopoulos, V., Wilson, R., and Irvine, K.D. (1997). Fringe modulates Notch-ligand interactions. Nature 387:908-913. Johnston, S. H., Rauskolb, C., Wilson, R., Prabhakaran, B., Irvine, K.D., and Vogt, T.F. (1997). A family of mammalian Fringe genes implicated in boundary determination and the Notch pathway, Development 124, 2245-2254. Kim, J., Irvine, K.D., and Carroll, S.B. (1995). Cell recognition, signal induction, and symmetrical gene activation at the dorsal/ventral boundary of the developing Drosophila wing. Cell 82: 795-802. Irvine, K.D. and Wieschaus, E. (1994). fringe, a boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79: 595-606. Irvine K.D. and Wieschaus E. (1994) Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes. Development. 1994 Apr;120(4):827-41.   Lab Support Cordelia Rauskolb, Assistant Professor, Research Vlad Panin, Post-doctoral Fellow Muriel Grammont, Post-doctoral Fellow Xiaofeng Liu, Graduate Student Liang Lei, Graduate Student Robert Major, Graduate Student Eunjoo Cho, Graduate Student Trudy Correia, Research Associate Irene Hao, Undergraduate Student