Date of Graduation

12-2013

Document Type

Dissertation (PhD)

Program Affiliation

Genes and Development

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Pierre D. McCrea, PhD

Committee Member

Michelle Barton, PhD

Committee Member

Fernando Cabral

Committee Member

Michael Galko

Committee Member

Malgorzata Kloc

Committee Member

Gregory May

Abstract

Plakophilin-3, the less studied member of the plakophilin-catenin subfamily, and the larger catenin family, binds directly to desmosomal cadherin cytoplasmic domains and enhances desmosome formation and stability. In mammals, plakophilin-3 is expressed at the highest levels in desmosome-enriched tissues such as epithelia, with the knock-out in mice producing corresponding reductions in ectodermal integrity. In tissue, cellular and intracellular contexts where plakophilin-3 is not at the desmosomal plaque, little is known about its functions in the cytoplasm or nucleus, where it also localizes.

My work employed embryos of the amphibian, Xenopus laevis, to examine plakophilin-3’s developmental roles. I first evaluated the expression pattern of endogenous plakophilin-3 mRNA and protein, revealing two principal and differentially expressed isoforms, and an enrichment in neural tissues as well as various epithelial structures. Further, despite conflicting reports on plakophilin-3’s nuclear localization, I showed that plakophilin-3 consistently localizes to the nucleus in both Xenopus naive ectoderm (animal caps) and mouse embryonic stem cells. To assess the in vivo functions of plakophilin-3, I employed sequence specific anti-sense morpholinos to knock-down its gene products. This resulted in developmental defects in the ectoderm, heart, the formation or maintenance of cilia, tactile sensation, neural crest establishment and migration, and the peripheral nervous system. These phenotypes were specific based upon the use of distinct non-overlapping morpholinos, and in the case of neural crest, the use of rescue experiments with morpholino-resistant plakophilin-3 mRNA. As noted below, the delineation of which phenotypes result from plakophilin-3's functions at the desmosome, or in the cytoplasm, versus or in addition to those in the nucleus, is a point of continuing interest. My results in all cases point to plakophilin-3’s essential roles in Xenopus development.

In probing for novel plakophilin-3 actions in the nucleus as well as elsewhere in the cell, I performed a yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with ETV1. ETV1 is an ETS transcription factor family member with important roles in neural development as well as human disease progression. Plakophilin-3’s interaction with ETV1 was confirmed using biochemical, functional, and developmental assays that centered ultimately upon plakophilin-3’s ability to regulate ETV1 transcriptional activity. These findings reveal the first sequence specific transcription factor to bind a member of the plakophilin-catenin subfamily.

Together, my work supports plakophilin-3’s essential roles in amphibian development, and shows plakophilin-3’s biochemical and functional association with ETV1. In a wider context, it strengthens the view that many (perhaps all?) catenin proteins play key roles at both cell-cell junctions and in the nucleus, as well as elsewhere in the cell, and raises the longer-term question of if such relationships might assist in integrating information across distinct cellular compartments.

Keywords

plakophilin, xenopus, catenin, cadherin, etv1, ets, wnt, desmosome, neural