Research Summary Developmental biology, segmentation of growing tissues, molecular genetics of Drosophila leg development Segmentation during Animal Development A fundamental process in the development of many organisms, including annelids, arthropods, and vertebrates, is segmentation. Segmentation serves to subdivide tissues into a series of repeating building blocks along either the body axis, or in some cases, the appendage axis, whereupon each basic unit can then be further elaborated upon during development. In most cases of segmentation, the subdivision of a tissue into repeating units must occur repeatedly as the tissue grows in size. The molecular mechanisms involved in generating a repeating segmental pattern in growing tissues are not well understood. We use Drosophila, with its powerful genetics and its known genome sequence, as a model organism in which to study the segmentation of growing tissues. In particular our work focuses on identifying the molecules required for segmentation of the Drosophila leg, a tissue in which segmentation must repeat continuously and must be coordinated with tissue growth. The Drosophila leg develops during larval stages from a cluster of undifferentiated cells, the leg imaginal disc. It is thus during larval stages of development that the leg tissue must be subdivided and borders made between different cell populations. Ultimately, adult Drosophila legs are composed of nine leg segments and each segment is separated from the next by a flexible joint. Notch signaling controls leg segmentation and growth In recent years some of the key members involved in Drosophila leg segmentation have been identified.  Importantly, the Notch signaling pathway plays an essential role in the segmentation and growth of the Drosophila leg. Notch is a transmembrane receptor protein.  There are two ligands for Notch in Drosophila, Serrate and Delta.  In addition, Fringe functions as a modulator of Notch signaling. Fringe inhibits a cell‚Äôs ability to respond to Serrate and potentiates a cell‚Äôs ability to respond to Delta.  The Notch signaling pathway is conserved amongst many animal species, and is fundamental to a wide range of developmental processes.  Furthermore, mutations in human Notch signaling components have been implicated in leukemia (TAN-1), stroke and dementia (CADASIL), and Alagille syndrome, a childhood syndrome resulting in chronic liver disease and segmentation defects.  Thus, results we obtain are likely to reveal developmental principles relevant to the biology of a wide variety of organisms.  Moreover, because Notch signaling also controls the growth of tissues, and unregulated growth is a key feature of cancer, identifying genes downstream of Notch signaling may provide insights into how tissue growth is controlled.  During Drosophila leg development, Serrate, Delta, and fringe are expressed in a segmentally repeated pattern.  At late stages of leg development they are expressed in a series of concentric rings within each of the future leg segments.  Through genetic manipulations, by removing or misexpressing Notch signaling components, we have shown that Notch signaling must be localized within each leg segment to promote the formation of boundaries (joints) that separate each leg segment and to induce leg growth.  This requirement for a segmentally repeated pattern of Notch activation highlights the importance in establishing appropriately patterned expression of the regulators of Notch activation.  Recently we have shown that the genes, homothorax, dachshund, and Distal-less, which are expressed in broad domains in the developing leg imaginal disc, are key regulators of the segmental pattern of Notch ligand and fringe expression.  Furthermore, we have identified two Serrate enhancers that respond to regulation by dachshund. Thus, a molecular pathway for segmentation of the Drosophila leg is beginning to be elucidated. However, the identities of the actual genes that execute segmentation remain largely unknown. The effectors of leg segmentation and growth Local activation of Notch in each leg segment has two important morphological consequences: leg segmentation and growth. Identifying the target genes regulated downstream of Notch signaling is crucial to our ultimate understanding of how, molecularly, leg segmentation and leg growth occur. We have identified several genes regulated by Notch activation that are themselves expressed in segmentally repeated patterns within developing legs. We are currently investigating how these genes contribute to Drosophila leg development. Four-jointed Four-jointed (fj) is regulated by Notch signaling in the leg, eye and wing of Drosophila and thus may be an important mediator of Notch function in a wide range of tissues.  Fj is expressed in a series of concentric rings in all segments of the developing leg disc and is required for the growth of many leg segments.  In addition, fj mutants lack a particular segment border, resulting in the fusion of two leg segments.  Fj encodes for a type-II transmembrane protein that can be cleaved to produce a signaling molecule.  In collaboration with Flora Katz (Texas A&M University), we have recently found that although fj expression is regulated downstream of Notch activation, fj also feeds back onto the Notch pathway by positively regulating the expression of the Notch ligands Serrate and Delta.  This adds an interesting complexity to the simple linear leg segmentation hierarchy (see Figure) and indicates that feedback loops may be an important means to precisely define where a segment border will form.  Given that fj is a signaling molecule, there is much interest in identifying the receptor and downstream components.  Together with Flora Katz, we have found that fj genetically interacts with abelson, enabled, and dachs, genes known and thought to affect the actin cytoskeleton.  Thus, one consequence of fj signaling may be an alteration in the actin cytoskeleton, which is consistent with the morphogenetic processes affected by fj (joint formation, which requires cell shape changes) in the leg and in other tissues.  We are currently testing candidate genes for whether they may act as the fj receptor. Odd-skipped Another downstream target gene is odd-skipped, whose expression is induced upon Notch activation.  The role of odd-skipped during leg development is currently unknown.  Odd-skipped is of special interest because odd-skipped is required for embryonic segmentation in Drosophila and is expressed in a segmentally repeated pattern in both embryos and in leg discs.  This suggests that odd-skipped may be a key gene in the segmentation of different tissue types.  To begin to address whether odd-skipped is an important downstream gene in leg segmentation, we made a construct that allows us to express odd-skipped ectopically in developing legs.  We are excited to have determined that ectopic expression in the leg induces ectopic sites of segmentation.  Thus, odd-skipped may be a key gene in effecting segmentation.  The making of joints requires changes in cell morphology.  Interestingly, ectopic odd-skipped expression can change the behavior of cells in the developing leg disc, which may be reflective of its role in segmentation.  Our goal now is to determine whether removal of odd-skipped during leg development has phenotypic consequences on leg segmentation, as we would predict.  There are three known cognate genes of odd-skipped in Drosophila, and we will determine their contribution to leg development as well. Selected Publications Rauskolb, C. (2001) The establishment of segmentation in the Drosophila leg. Development 128:4511-21. Irvine K.D., and Rauskolb, C. (2001) Boundaries in development: formation and function. Annu Rev Cell Dev Biol. 17:189-214. Download PDF. Buckles, G. R., Rauskolb, C., Villano, J. and Katz, F. N. (2001). Four-jointed interacts with dachs, abelson, and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg. Development 128:3533-42. Rauskolb, C., Correia, T. and Irvine, K. D. (1999). Fringe-dependent separation of dorsal and ventral cells in the Drosophila wing. Nature 401:476-480. Rauskolb, C. and Irvine, K. D. (1999). Notch-mediated segmentation and growth control of the Drosophila leg. Dev. Biol. 210:339-350. Papayannopoulos, V., Tomlinson, A., Panin, V. M., Rauskolb, C. and Irvine, K. D. (1998). Dorsal-ventral signaling in the Drosophila eye. Science 281:2031-2034. Lab Members Olga Dunaevsky, Research Technician