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Control of Tissue Patterning and Growth During Development
Summary: Kenneth Irvine studies molecular mechanisms that regulate the growth and shape of tissues during animal 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 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 reexamine the influence of long-range morphogens on Drosophila wing growth. For example, although Decapentaplegic (DPP), a member of the TGFβ (transforming growth factor-β) 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. Our results support a class of models that 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. Our current experiments are focused on defining the molecular mechanism by which the DPP morphogen gradient influences wing growth in Drosophila.
The Fat Signaling Pathway
In a complementary 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 the Drosophila gene fat, which encodes a transmembrane receptor for this pathway. Fat signaling influences gene expression, tissue growth, and planar cell polarity. Defining the molecular mechanisms by which Fat signaling influences these processes is a major focus of our research.
One product of this research has been the identification and characterization of the dachs gene as a critical downstream effector of Fat signaling. Although 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 clue to the role of Dachs has come from the 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 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.
Genetic studies have allowed us to identify additional components of the Fat signaling pathway and to characterize their functional relationships. We identified the kinases Discs overgrown and Warts as components of a Fat signaling pathway by showing that they regulate a common set of downstream genes in multiple tissues, including wingless, Serrate, four-jointed, Diap1, cyclinE, and expanded. Genetic experiments positioned the action of discs overgrown (dco) upstream of dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs, and dco in vivo, consistent with the placement of Warts as a downstream component of the pathway. The tumor suppressors Merlin, expanded, hippo, salvador, and mats also share multiple Fat pathway phenotypes but appear to regulate Warts activity in parallel to Fat, through the Hippo signaling pathway. Our results have functionally linked what had been four disparate groups of Drosophila tumor suppressors, established a basic framework for Fat signaling from receptor to transcription factor, and implicated Warts as an integrator of multiple growth control signals. We are continuing to decipher the biochemical basis for Fat signaling and to investigate whether an analogous Fat signaling pathway exists in mammalian cells.
We have also explored the relationship between Fat and Expanded, which act as upstream regulators of Warts within the Fat and Hippo pathways, respectively. Mutations in either expanded or fat can be rescued to viability simply by overexpressing Warts, indicating that their essential function is their influence on Warts, rather than reported effects on endocytosis or other pathways. These rescue experiments also separated the transcriptional from the planar cell polarity branches of Fat signaling and revealed that Expanded does not directly affect planar cell polarity. We also investigated the genetic relationship between expanded and fat, and showed, contrary to other reports, that they have additive effects on imaginal disc growth and development. Our observations argue against recent proposals that Fat acts as a receptor for the Hippo signaling pathway and instead support the proposal that Fat and Expanded act in parallel to regulate Warts through distinct mechanisms.
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 the influence of 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 β1,3-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.
Our research is also supported by a grant from the National Institutes of Health.
Last updated: January 4, 2008
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