Control of Tissue Patterning and Growth During Development Research in my laboratory is directed toward understanding the regulation and coordination of tissue patterning, growth and morphogenesis during animal development. Much of our research takes advantage of the powerful genetic, molecular, and cellular techniques available in Drosophila melanogaster, which facilitate both gene discovery and the analysis of gene function.  Patterning and growth during development Understanding how growth is controlled is a major goal of developmental biology. Decades ago, regeneration experiments revealed an intimate relationship between tissue patterning and tissue growth, but the molecular basis for this relationship has remained elusive. We are currently engaged in projects whose long term goal is to define relationships between patterning and growth in developing tissues. One approach we have taken is to re-examine the influence of long range morphogens on Drosophila wing growth. For example, while Decapentaplegic (DPP), a member of the TGFß family, has long been known to be important for wing growth, how it actually influences growth had remained unclear. We have re-examined this by using a new approach for regulating gene expression in Drosophila, which enabled us to exercise quantitative and temporal control over expression of transgenes in clones of cells. Our results support a class of models which posit that growth is regulated by the slope of morphogen gradients. We found that the juxtaposition of cells that perceive different levels of DPP signaling is essential for cell proliferation in parts of the wing, and can be sufficient to promote the proliferation of cells throughout the entire wing. These observations provided the first direct demonstration that the slope of a morphogen gradient can regulate growth during development.  The Fat signaling pathway In a second approach to investigating the relationship between developmental patterning and growth, we have been investigating a new signaling pathway that links these processes. We refer to this pathway as the Fat signaling pathway, after a Drosophila gene called fat, which encodes a transmembrane receptor for this pathway. Fat signaling influences at least three processes during Drosophila development: it influences a form of cell polarity (planar cell polarity), it influences gene expression, and it influences growth. Defining the molecular mechanisms by which Fat signaling influences gene expression and growth is currently a major focus of research in the lab. One product of this research has been the identification and characterization of the dachs gene as a critical downstream effector of Fat signaling. While dachs was first identified almost a century ago, it had been relatively little studied. We found that mutation of dachs greatly reduces the growth of legs and wings in Drosophila, dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells, and dachs mutations completely suppress the effects of fat mutations on gene expression and growth. A intriguing clue to the role of Dachs has come from observation that Dachs protein localization is influenced by Fat signaling. Fat is regulated by two proteins, Dachsous, and Four-jointed, which are expressed in gradients in developing tissues. Dachs protein is normally asymmetrically localized in the developing wing, with higher levels on the distal side of each cell. Manipulations of Dachsous and Four-jointed expression suggest that this asymmetry is directed by the Dachsous and Four-jointed expression gradients. This observation provides a basis for beginning to understand how a gradient of protein expression might be converted into a constant signal across a field of cells. Other work in progress has allowed us to identify additional components of the Fat signaling pathway, and we are also beginning experiments to investigate Fat signaling in mammalian cells.  Notch signaling Over the last several years, the regulation and functions of the Notch signaling pathway has been a major focus of research in the lab. Notch is a receptor protein that mediates a wide range of cell fate decisions during animal development. In humans, aberrant Notch signaling has been linked to leukemia (TAN-1), and congenital syndromes associated with stroke and dementia (CADASIL), and liver, cardiovascular, and skeletal defects (Alagille, spondylocostal dysostosis). The Notch receptor and its ligands are modified by an unusual form of glycosylation, which is initiated by the attachment of fucose to Serines or Threonines within epidermal growth factor-like (EGF) repeats. We have studied the influence of this post-translational modification using a combination of Drosophila genetics, cell culture, and biochemistry. Protein O-fucosyltransferase 1 (OFUT1), the enzyme that initiates the synthesis of O-linked fucose, acts both as a fucosyltransferase to modify the Notch receptor, and as a chaperone to promote Notch receptor folding. Fringe is a glycosyltransferase that modifies the O-linked fucose on Notch by addition of ß1,3 linked N-acetylglucosamine. This further glycosylation of Notch both inhibits the binding of one ligand, Serrate, to Notch and potentiates the binding of another ligand, Delta, to Notch. By reproducing the influence of glycosylation on ligand binding in vitro with purified components, we have been able to demonstrate that the simple addition of N-acetylglucosamine to Notch is sufficient to alter the interaction of Notch with its ligands. Notch signaling is required for an enormous number cell fate decisions in metazoans, but Fringe is only required for a subset of Notch signaling events. We have taken advantage of this over the years to identify and characterize requirements for Notch signaling in different tissues. Most recently we have focused on a critical yet poorly understood role for Notch in the subdivision of the developing Drosophila wing into distinct groups of cells that do not intermix, called compartments. Because Notch has been implicated in separating cells in a wide variety of contexts, from somitogenesis and brain compartmentalization in vertebrates to leg and body segmentation in insects, we think that the results of these studies will be broadly relevant. Our characterization of the role of Notch has led us to propose that it effects compartmentalization in a novel way: rather than establishing distinct cell affinities, it induces a property or behavior of cells at the border that prevents them from intermixing, which we refer to as a fence. In characterizing the cellular and molecular basis for this Notch-dependent cell separation, we discovered a distinct, Notch-dependent organization of the actin cytoskeleton, and identified requirements for F-actin and Myosin.  Developmental Glycobiology There are an increasing number of examples in which post-translational modification of proteins by glycosylation plays important roles in regulating their activity, but the requirements for some forms of glycoylation remain poorly understood. In order to identify and characterize additional requirements for glycosylation, we have conducted genetic and biochemical studies in Drosophila on several genes predicted to encode glycosyltransferases, including  sialyltransferase, b1,4-galactosyltransferases, and fucosyltransferases. Currently we are focusing on b1,3 galactosyltransferases that we have found are essential for viability in Drosophila. One gene of interest is closely related to mammalian core 1 b1,3 galactosyltransferase, which transfers Galactose onto O-linked GalNAc. This is of particular interest because alterations in O-GalNAc glycans have been correlated with tumor metastasis. We have identified a Drosophila core 1 b1,3 galactosyltransferase that is essential for morphogenesis, and we are currently characterizing its requirements in detail.