Control of Tissue 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 engaged in projects whose long-term goal is to define relationships between patterning and growth in developing tissues. 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 One approach we have taken to elucidate the relationship between patterning and growth is to examine the influence of long-range morphogens on Drosophila wing growth. For example, although Decapentaplegic (DPP), a member of the TGF? (transforming growth factor-?) family, has long been known to be important for wing growth, how it actually influences growth had remained unclear. To reexamine this, we used 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. We found that the juxtaposition of cells that perceive different levels of DPP signaling can influence cell proliferation in the developing Drosophila wing. Our results supported a class of models that posit that growth is regulated by the slope of morphogen gradients. More recently, we have made progress toward elucidating the molecular mechanisms that link the DPP morphogen gradient to wing growth. We found that DPP signaling acts through the Fat-Warts-Hippo signaling network. DPP signaling regulates the expression and localization of Fat pathway components; Fat signaling, through its downstream effector Dachs, is required for the influence of the DPP gradient on cell proliferation. Moreover, juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat pathway target genes and cell proliferation, whereas uniform expression of four-jointed and dachsous inhibits cell proliferation. Our observations identified Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth.  The Fat Signaling Pathway In addition to characterizing the role of Fat signaling in morphogen-dependent growth and patterning, we are also engaged in projects to define the molecular basis for Fat signal transduction. The Fat signaling pathway in Drosophila is named for a Drosophila gene, fat, which encodes a cadherin protein that acts as a transmembrane receptor for this pathway. Fat signaling influences both gene expression and planar cell polarity. We discovered that the influence of Fat signaling on gene expression is effected through an intersection with the Hippo-Warts signaling pathway. Several components of this pathway act as tumor suppressors or oncogenes, both in Drosophila and in mammals. Our studies have analyzed multiple steps of Fat signaling, from regulation of the Fat receptor at the membrane, to the intersection with Warts-Hippo signaling, to the regulation of gene expression through the transcriptional coactivator Yorkie. Genetic studies in Drosophila identified the four-jointed gene as a regulator of Fat signaling. We recently found that four-jointed encodes a protein kinase that phosphorylates Ser or Thr residues within cadherin domains of Fat and its transmembrane ligand, Dachsous. Four-jointed functions in the Golgi to phosphorylate these domains of Fat and Dachsous, which will be extracellular, and is the first molecularly defined Golgi kinase. The phosphorylation of Fat and Dachsous appears to influence interactions between them. Characterization of Four-jointed has also led us to the identification of other novel Golgi kinases, which we are investigating. The dachs gene occupies a central position within the Fat signaling pathway, as dachs influences both the transcriptional and planar cell polarity outputs of Fat signaling. Dachs encodes a myosin-related protein, and 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 have revealed 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 signal across a field of cells. Yorkie, a transcription factor of the Fat and Hippo signaling pathways, is negatively regulated by the Warts kinase. We used a recent technology for characterization of phosphorylated proteins (Phos-tag gels) to characterize Warts-dependent phosphorylation of Yorkie in vivo and to show that Warts promotes phosphorylation of Yorkie at multiple sites. We also observed that Warts inhibits Yorkie nuclear localization in vivo. We identified serine 168 as a critical site through which negative regulation of Yorkie by Warts-mediated phosphorylation occurs, but this site was not sufficient to explain effects of Hippo signaling on Yorkie in vivo, and we have since identified two additional Warts phosphorylation sites. Many components of Fat signaling are conserved, but the mammalian Fat signaling pathway has not yet been well characterized. To facilitate characterization of the roles of Fat signaling in mammals, we created and have begun to analyze a gene-targeted mutation in a murine Dachsous homolog.  Notch Signaling 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); 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 (EGF)-like repeats. We have used a combination of Drosophila genetics, cell culture, and biochemistry to study this post-translational modification. 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 ?1,3-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 demonstrated that the simple addition of N-acetylglucosamine to Notch is sufficient to alter the interaction of Notch with its ligands. We are continuing to try to define the structural basis for this influence of glycosylation on Notch-ligand interactions.