Researcher(s)
- Paige Heeter, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Erica Selva, Biological Sciences, University of Delaware
Abstract
The canonical Wnt cell signaling pathway contributes to embryonic development as well
as cell upkeep in mature individuals. When this pathway is disrupted during development, many
different defects can occur, and in adults, Wnt pathway disruption is a major factor behind
cancers. Currently, much is known about the molecular events required for signaling in receiving
cells; however, there are still questions that remain regarding our understanding of what occurs
in the cells that produce and deliver the Wnt signal. The maturation of virtually all Wnt ligands
within signal-sending cells is designated to highly conserved yet understudied components,
Porcupine (Por) and Wntless (Wls). Proper maturation of the Wnt signaling molecule is vital for
downstream function, but to study the requirements for functional Wnt maturation in signal
sending cells, reliable assays are needed to assess Wnt maturation, secretion, and its ability to
activate the pathway in receiving cells. My summer research focused on optimizing pathway
activation in receiving cells. Previous research relied on autoactivation assays in which signaling
cells also acted as receiving cells; however, results of these experiments did not provide
sufficient activation results. In the autoactivation assay, only β-catenin accumulation can be
accessed rather than the Wnt Top flash reporter assay. We attempt to develop a transactivation
assay in which separate cell groups act as signaling and receiving cells. Transfected signaling
cells were mixed with transfected receiving cells, and activation levels were assessed via
Western blots. Activation of the Wnt pathway is roughly twofold increase in β-catenin protein
levels relative to control cells. However, we were not able to detect the Top flash activity through
Firefly luciferase expression in Western blots. In addition to optimizing our transfection
efficiency, we are currently working to observe Firefly luciferase levels in the blots and
eventually move on to luciferase plate reader assays.