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.