Investigating the localization and self-organization of core planar cell polarity proteins using an endogenously tagged Fz6 fusion protein

Abstract

The planar cell polarity (PCP) pathway directs the polarity of epithelia and other planar tissues across animal phyla. In humans, the PCP pathway regulates hair follicle development and wound healing in the epidermis. Significantly, recent evidence has suggested that in mice, the core PCP protein Fz6 does not have an asymmetric localization in the epidermis, contrary to prevailing models. Critically, this evidence was produced in a system with multiple confounds, so this result must be clarified. On a more fundamental level, there is present ambiguity surrounding the nuanced process by which PCP proteins interact to establish their polarity. In this thesis, I report on the development and characterization of an endogenously tagged Fz6-3xGFP mouse line and cell line which are very useful for investigating these two areas of ambiguity. With live imaging and immunohistochemistry, I demonstrate that this protein can be visualized live in vivo and localizes normally over time both in vivo and in cell culture. Further, I utilize this mouse line along with a mosaic cell culture technique and extended resolution confocal microscopy to provide preliminary evidence that Fz6 has a unipolar localization, contradicting the recent report. Lastly, I report on the limitations of and alternatives to a micropatterning approach that may be eventually useful in combination with this cell line to clarify the mechanism of PCP establishment, but currently does not permit long term cell survival. Ultimately, this project describes the first endogenously tagged PCP protein which has applications in diverse future studies of the PCP pathway. I provide future directions that could expand on preliminary results provided in this report to clarify open questions relating to PCP.