Drosophila as a model organism | Tissue patterning | Tissue sculpting | Tissue growth
Drosophila as a model organismDrosophila offers a number of advantages that have made it one of the best model organisms for genetic studies of development and disease. These include a rapid life cycle, relatively simple genetics (with just four chromosome pairs), and a host of powerful genetic tools and techniques developed over the last century that make it possible to activate or inactivate genes in specific tissues and at specific times, and evaluate the consequences.
Despite our differences, many of the fundamental cellular processes that drive fly development are conserved in humans. This is reflected at the level of the two genomes, with humans and Drosophila sharing many genes in common. In fact, approximately two-thirds of human disease genes have clearly identifiable counterparts in flies. Genetic studies in flies have played a central role in our understanding of how the products of many of these genes function normally and how their misregulation can cause disease.
Much of our work focuses on imaginal discs. These are a series of small epithelial sacs that are formed in the embryo and grow rapidly throughout the four days of larval life. Once fully grown, the imaginal discs undergo extensive unfolding and rearrangement to form many of the adult body structures, including eyes, wings, and legs. Because of their simple structure, rapid growth, and easy accessibility, the imaginal discs are an ideal system for studying how growth and organization of epithelial tissues are controlled.
Tissue patterning: Control of Hedgehog signaling by G-protein-coupled receptor kinasesAs developing tissues grow, they also acquire a form or pattern. This involves subdividing the cells into discrete populations that will form the various structures found in the fully mature tissues. This subdivision is controlled by proteins called morphogens which are secreted from groups of cells at specific locations and travel through tissues to control the fates of cells some distance away.
One important class of morphogens controlling both the growth and patterning of most tissues in mammals and flies are the Hedghehog (Hh) proteins. Binding of Hh to its receptor Patched on the cell surface triggers the accumulation and activation of a seven-pass transmembrane protein called Smoothened. Smoothened in turn activates an intracellular signaling pathway that controls the accumulation and nuclear entry of the transcription factor Ci. When Hh signaling is active, Ci is stabilized and can activate expression of Hh target genes.
In the fly wing disc, Hh signaling activates target genes in a central stripe of cells. These cells will go on to form the central region of the adult wing. We have been characterizing animals lacking a kinase, called Gprk2. Gprk2 mutants show a loss of the central region of the wing, which is a defect characteristic of mutations in genes required for cells to respond to the Hh signal.
We have shown that Gprk2 regulates Smoothened phosphorylation, internalization from the cell surface, and turnover (Cheng et al., 2010). Surprisingly, despite the fact that they lose Hh target gene expression, Gprk2 mutants show signs typical of activation of the signaling pathway, including excessive accumulation of both Smo and Ci. We are currently trying to understand precisely how Gprk2 regulates Smoothened and why pathway activation fails to induce Hh target gene expression in the absence of this kinase.
Hh proteins in mammals play important roles during development and in stem cell maintenance in adults. Misregulation of Hh signaling is a known cause of certain congenital malformations and of cancers, including medulloblastoma and basal cell carcinoma. Our studies in flies will thus have implications for how the activity of this pathway is regulated in mammals, both in normal tissue homoeostasis and disease.
Tissue sculpting: Shaping cells and tissues through dynamic control of actin cytoskeleton regulatorsRegulation of cell shapes is an important mechanism for generating the complex forms of animal tissues. Dynamic regulation of the actin cytoskeleton, a sort of cellular scaffold composed of filaments of the protein actin, is a conserved and major driving force for reshaping epithelial cells and tissues. Tensional forces in cells are generated by the pulling action of Myosin proteins on actin filaments. Through the anchoring of actin filaments to various protein complexes associated with cellular membranes, the forces applied to actin filaments can be translated into deformations of the cell surface that change cell shapes.
We have been characterizing the functions of two kinases that are necessary for controlling epithelial cell shape in flies. The first is a kinase we called Drak (Neubueser & Hipfner, 2010). Drak controls cytoskeletal tension by regulating the phosphorylation of the non-muscle Myosin II regulatory subunit (MRLC). In its phosphorylated form, MRLC stimulates actin-myosin contraction. In tissues from animals lacking Drak, we found that MRLC phosphorylation was abnormally low. MRLC phosphorylation was decreased even further when the levels of a partially redundant MRLC kinase, Rok, were simultaneously reduced. As a consequence, animals lacking Drak or both Drak and Rok display significant malformations in many epithelial tissues. Using genetic and biochemical approaches, as well as time-lapse video microscopy, we are trying to determine precisely where and how these kinases control MRLC phosphorylation.
The second kinase is one we called Slik (Hipfner et al., 2004). We showed that Slik is required for the phosphorylation of a protein called Moesin. In its active, phosphorylated form, Moesin acts as a cross-linker to connect actin filaments to proteins at the cell surface. As a result of the failure of Moesin activation in animals lacking Slik, these animals display malformation of epithelial tissues, including imaginal discs. Similarly, in the absence of either of these proteins, light-sensing photoreceptor cells in the eye are malformed, impairing their ability to respond normally to light. Using candidate and screening approaches, we are characterizing the signaling network that regulates Moesin activation, and thus epithelial cell shape, via Slik.
Both Drak and Slik have clear counterparts in humans. Therefore, we expect that our analyses of the functions of these two kinases in flies will provide important insights into how the human kinases function. In doing so, we may gain a better understanding of how these kinases and their substrates contribute to diseases where the organization of epithelial-derived tissues is disturbed, including cancer and certain forms of congenital blindness and deafness.
Tissue growthIn addition to its role in regulating Moesin activation, Slik also controls tissue growth in the developing imaginal discs. We showed that overexpression of the kinase increases the rate at which cell proliferate, leading to tissue overgrowth. Animals lacking Slik display the opposite phenotype, with signs of reduced cell proliferation and a dramatic growth delay (Hipfner et al., 2003). The ability of Slik to drive cell proliferation is independent of its ability to regulate Moesin. Instead, we could show genetically that Slik-driven tissue growth depends upon the activity of another kinase, Raf, which has a well-known function in regulating tissue growth in flies and tumourigenesis in mammals. Using a variety of approaches, we are trying to determine the molecular basis for the genetic interaction between Slik and Raf in promoting cell proliferation and tissue growth.