Douglas A. Harrison - US grants

Harrison 9723944 The coordinated proliferation and differentiation of distinct cell types is critical to the proper development and maintenance of any multicellular organism. For this coordination to occur, cells must have a means to communicate with one another. The broad long-term objectives of this research are to elucidate mechanisms of cell-cell signaling and to understand how different signals are interpreted to regulate developmental events. Toward these goals, the Janus kinase (JAK) signaling pathway will be studied in Drosophila melanogaster. This system will be used because it is developmentally important, relatively simple, and is conserved between flies and mammals. In mammals, the pathway is know to be comprised of the non-receptor tyrosine kinases (JAKs) and the signal transducers and activators of transcription (STATs). In flies, one JAK (hopscotch) and one STAT (marelle) gene have been recently identified. The genetic and developmental manipulations available in Drosophila make it an ideal model system to further study JAK/STAT signaling. These approaches will complement and extend the biochemical analyses of the pathway conducted in mammalian cells. Mutational analysis has already uncovered a new component of JAK/STAT signaling in Drosophila, unpaired (upd). An understanding of the involvement of upd in JAK signaling is central to the research proposed here. Other molecules integral to the JAK pathway, or that strongly interact with components of the pathway, will be identified and characterized using the genetic tools available in Drosophila. The specific goals of this research are to: 1) Characterize the unpaired locus at the molecular level 2) Determine the functions of the Upd product in JAK signaling 3) Characterize the roles of Upd function in Drosophila development 4) Identify other components of and factors interacting with the JAK pathway

0091535
Harrison

Communication between cells is a vital factor in proper development and maintenance of multicellular organisms. The number of molecules that serve as signals is enormous, yet the mechanisms by which cells can respond to signals are much more restricted. A finite number of signaling pathways have emerged as reutilized intracellular signaling cascades that initiate appropriate cellular responses. The long term objectives of this research are to elucidate mechanisms of cellular communication and to understand how different signals are integrated and interpreted to regulate developmental events. One conserved intracellular cascade that has been found in organisms from slime mold to human is the Janus kinase (JAK) signaling pathway. In vertebrates, JAK signaling is the primary mechanism for response of cells to a wide array of cytokines and growth factors. The fruit fly, Drosophila melanogaster, was chosen to study JAK signaling because it uses the JAK pathway in the same manner as humans, yet is amenable to genetic and developmental manipulation. The only known activator of JAK signaling in Drosophila is the Unpaired (Upd) protein. Previously funded research (IBN-9723944) focused on the molecular and biochemical characterization of upd. The Upd protein bears no recognizable similarity to previously identified mammalian proteins, but is similar to a protein from a closely related fruit fly species, Drosophila ananassae. Both Om(1E) and updgenes are found in both of these Drosophila species. Furthermore, the recent release of the Drosophila genome sequence has identified two additional, less conserved predicted proteins that have homology to Upd. The functions of these three new genes will be investigated, as well as the relationships of these genes to upd and to one another. Understanding of the molecules and mechanisms of JAK pathway activation in Drosophila will likely provide insights into developmental signaling in many multicellular organisms. The primary goals of this research are to: (1) characterize, molecularly, biochemically, and phenotypically, the Drosophila melanogaster Om(1E) gene, (2) determine the functional relationships between Om(1E) and upd, (3) characterize, molecularly and phenotypically, two other proteins with homology to Upd, and (4) identify additional components of JAK signaling and related pathways using genetic screens.

0318776
Harrison

A small number of defined signaling pathways have emerged as reutilized intracellular signaling cascades that initiate cellular responses. One of these is the Janus kinase (JAK) signaling pathway. JAK signaling is the primary component of the response of many vertebrate cells to a many cytokines and growth factors. Consequently, the JAK pathway is essential for a many developmental events, including hematopoiesis, immune system development, and general growth. The pathway has been evolutionarily conserved from insects to human. Study of JAK signaling in the fruitfly, Drosophila melanogaster, is particularly attractive because it is amenable to genetic and developmental manipulation and is much simpler than the vertebrate counterpart, with only single representatives of each type of pathway molecule. As in vertebrates, the pathway is critical to many developmental events. Previously funded NSF activities have uncovered a requirement for JAK signaling in the patterning of the follicular epithelium, the monolayer of somatic cells that covers the developing egg and forms the eggshell and specialized structures. The follicular epithelium is comprised of 5 different cell fates that are determined by their position along the anterior-posterior axis The JAK ligand, Upd, as well as a homologous protein, are secreted from the poles of the egg chamber and a gradient of JAK pathway activity is created that is highest at the anterior and posterior termini. The gradient of JAK pathway activity determines the fates of the follicular cells. This suggests that Upd may act as a classical morphogen, a molecule that patterns a field of tissue into different cell fates that are assigned by virtue of position relative to the morphogen source.

The two primary goals of this research will address the hypothesis that Upd may act as a morphogen. First, the mechanisms by which a gradient of JAK pathway activity is established in the follicular epithelium will be determined. The distribution of the Upd and homologous proteins their contributions to activation of the JAK pathway and establishment of epithelial pattern will be assessed. Second, the mechanisms by which the Upd ligand is transported to receiving cells will be examined. Known morphogens are distributed by passive diffusion, active endocytic transport, or a combination of the two. The contributions of these mechanisms to distribution of Upd and homologues will be examined.

The proposed research will be carried out primarily as the major training activity of graduate students. Additional roles will be played by undergraduates seeking learn about biology through conducting independent research projects. As in the past, the majority of students involved in these research activities are likely to be women and/or Kentucky natives. Results of the proposed activities will be presented at professional meetings by the students who carry out the research.

The development of multicellular animals from a single-celled fertilized egg is a complex process that requires extensive communication between enormous numbers of cells. It is the long-term goal of this research to better understand how cellular signaling is used to direct activities that sculpt a multicellular organism. The approach taken in this project is to study the mechanisms by which one evolutionarily conserved signal cascade, the Janus kinase (JAK) signaling pathway, regulates development in the fruit fly, Drosophila melanogaster, as a model for developmental cell signaling in other animals, including humans. Study of JAK signaling in fruit flies is particularly attractive because it is amenable to genetic and developmental manipulation and because only the three Unpaired (Upd) family proteins are believed to act as signals for the JAK pathway. Previous studies have examined the developmental functions of Upd and Upd2. The current project will use genetic, developmental, and biochemical methods to determine the functions of Upd3 in development and JAK signaling, as well as to determine the mechanisms by which the Unpaired family proteins act together to regulate JAK signaling during development. It is expected that mechanisms uncovered in this system will be applicable to multi-gene families of cell signaling proteins found in other animals. This project will provide opportunities for the education and training of young biologists and will be the primary training activity of graduate students in the laboratory, with additional roles played by undergraduates seeking to learn about biology through conducting independent research projects.