Molecular Mechanisms Underlying Cytoneme Formation by Sonic Hedgehog-Producing Cells

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ABSTRACT Proper physiological development relies on cell-to-cell communication via secreted morphogens. These potent signaling molecules signal at both short and long range in temporal- and tissue-specific manners to determine cell fate and pattern tissues. Sonic Hedgehog (SHH), a well-studied vertebrate morphogen, plays an integral role in a variety of developmental processes including neural tube patterning and limb development. Defects in the pathway result in developmental malformations, while aberrant activation has been associated with developmental disease and malignant transformation. SHH-producing cells extend thin, actin-based projections to transport SHH to receiving cells. These specialized filopodia are termed “cytonemes” due to their thread-like appearance. Cytonemes are proposed to play a key role in tightly regulating morphogen concentration gradients during development. However, little is known about the molecular mechanisms driving cytoneme formation. This is mostly due to the difficulty to study the delicate structures using conventional fixation methods. To permit mechanistic studies in vitro, our lab developed a modified electron microscopy fixative (MEM- fix) protocol that preserves cytoneme integrity for confocal analysis. Using MEM-fix, along with a combination of advanced imaging and biochemical techniques, our lab has found that expression of SHH can promote cytoneme initiation in cultured murine fibroblasts. Furthermore, we identified a requirement for the SHH coreceptors and adhesion proteins, Cell adhesion-associated, Down- regulated by Oncogenes (CDON) and Brother of CDON (BOC), along with Dispatched (DISP) deployment receptor in SHH-mediated cytoneme formation and stability. These findings along with identification of interactions between SHH, DISP, and BOC as well as DISP and CDON point towards cytoneme-initiating signaling occurring downstream of SHH binding to these transmembrane proteins. However, the specific interactions and intracellular signaling pathways that initiate cytoneme outgrowth have yet to be identified. My proposed project is focused on determining the SHH-activated signals occurring in response to its association with BOC and DISP that drive cytoneme initiation in SHH- producing cells. The first goal of my project is to elucidate the specific cell surface interactions between DISP, BOC, CDON, and SHH that signal to intracellular cytoskeletal regulators. The second aspect of my project is to elucidate the regulatory molecules that directly promote actin nucleation and polymerization in response to SHH. To address these outstanding questions, I am combining targeted genetic and biochemical techniques with advanced imaging approaches to methodically interrogate the signal cascade resulting in SHH-mediated induction of cytoneme formation. I will confirm findings in vivo by using our recently developed protocol facilitating imaging of cytonemes in mouse embryos.