Investigating the Role of Dispatched in Hedgehog Ligand Transport and Delivery
During the development of all metazoans, the Hedgehog (Hh) signaling pathway provides instructional cues influencing a variety of cellular processes. The pathway ligand, Hh, is dually lipidated by cholesterol and palmitate, which effectively anchors the molecule to the lipid bilayer of the signal producing cell. To complicate the Hh pathway induction process, the Hh ligand is often produced at a significant distance from the cells it influences. Only one known conserved molecule, Dispatched (Disp), can alleviate the membrane tethering imparted by Hh lipidation. Underscoring the importance of Disp protein during development, knockout animals succumb to lethality at E9.5, an exact phenocopy of the knockout of the essential signal transducer of the pathway: Smoothened. Furthermore, mutations within Disp have been found in patients with Holoprosencephaly, the most common cause of human forebrain malformations, which is frequently caused by inhibition of Hh signaling during development. Very little is known regarding the functional or regulatory mechanisms enabling Disp to transport and release Hh ligand. This dissertation aimed to narrow this gap and began with investigation into the role of Disp in a largely ignored mechanism of Hh ligand transport known as cytoneme-mediated morphogen transport. This method of morphogen transport utilizes fragile, thin cytoplasmic extensions which deliver cargo directly from the source of production to ligand responding cells. Through the use of a modified electron microscopy fixative, which we named MEM-fix, to maintain cytonemes for traditional cell biological analysis, I established an in vitro cytoneme system capable of modeling in vivo cytoneme biology. This in vitro cytoneme system uses Schneider 2 cells, an embryonically derived Drosphila cell line, which demonstrate competency to produce and utilize cytonemes as a mechanism of Hh transport. Equipped with this tool, I investigated the relationship between cytonemes and Hh pathway components. In doing so, I uncovered a previously unknown Disp requirement in cytoneme-mediated transport of Hh ligand and subsequently a Disp-mediated cytoneme stabilizing effect. Through these studies into Disp-mediated cytoneme transport, I identified a Disp cleavage event of Disp that regulates the ability to release Hh ligand. The second part of this dissertation details a collaborative effort in which we discovered that the Furin family of proprotein convertases facilitate the cleavage of Disp at a conserved site of the first extracellular loop. The Furin family has been implicated in cleaving ligands and receptors of other developmental signaling pathways, but no reports exist linking the Furin family to regulation of the Hh pathway. Therefore, we investigated the functional consequence of this Disp cleavage event, and our results suggest that this cleavage likely influences the proper trafficking of Hh for efficient release both in vitro and in vivo. To our knowledge, this is the first report of a regulatory protein partner controlling the activity of Disp in releasing Hh.
Cytonemes, Dispatched, Hedgehog Signaling, MEM-fix, Morphogen, Morphogen Transport, Chemicals and Drugs, Genetic Processes, Medical Biochemistry, Medical Cell Biology, Medical Sciences, Medicine and Health Sciences
University of Tennessee Health Science Center
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