Mammalian embryonic development is a highly stereotyped and coordinated process. Although tremendous diversity exists across species, the fundamental processes that drive spatiotemporal patterning and shaping are regulated by only a few signaling pathways. Wnt signaling is one such pathway that is involved in many aspects and stages of development. The many modalities of Wnt signaling, initiated through different ligand receptor combinations, elicit unique molecular and cellular responses, which historically have been differentiated by their ability to impact either tissue specification or tissue morphogenesis. In this paradigm, canonical Wnt signaling, or Wnt/β-catenin signaling, utilizes the transcriptional co-activator β-catenin to affect gene transcription and cell proliferation, thereby impacting tissue specification. In contrast, non-canonical Wnt signaling relies on β-catenin-independent mechanisms to affect cell migration to drive tissue morphogenesis. While the mechanisms and outputs of canonical Wnt signaling are well-studied in many physiological and pathological contexts, a reciprocal understanding of the molecular and cellular mechanisms governing non-canonical Wnt-driven tissue morphogenesis has remained limited. This dissertation seeks to augment our understanding of the molecular and cellular mechanisms by which one major non-canonical Wnt pathway – Wnt5a-Ror signaling, defined as the signaling cascade initiated by Wnt5a ligands and Ror receptors – regulates embryonic development and how its misregulation can lead to congenital birth defects. This is achieved through further delineation of the Wnt5a-Ror signaling pathway based on the identification and characterization of a downstream regulatory component, the E3 ubiquitin ligase Pdzrn3, in addition to assessment of the roles of Dishevelled (Dvl) scaffolding proteins as they mediate both Wnt5a-Ror and Wnt/β-catenin signaling pathways in physiological as well as pathological contexts.
In Chapter 1, I briefly overview the history of Wnt signaling as a major developmental signaling network involved in a variety of unique biological processes. I detail the morphogenesis changes associated with misregulation of Wnt5a-Ror signaling in key model organisms, namely the widened and shortened of the body axis, head, tail and limbs. I also discuss the similarity between these Wnt5a-Ror signaling mutant organisms and Robinow syndrome (RS) human patients, which share many overlapping physical features and possess mutations in components of Wnt5a-Ror signaling. Finally, I delve more specifically into RS mutations that occur in DVLs, a family of evolutionarily conserved scaffolding proteins involved in facilitating multiple Wnt signaling pathways and indicate key questions surrounding the function and downstream changes driven by these proteins.
In Chapter 2, I focus on augmenting our mechanistic understanding of Wnt5a-Ror signaling that occurs in normal physiological contexts. In this team effort, I and others use a whole-cell proteomics approach to identify downstream effectors of Wnt5a-Ror signaling, and through this approach discover the E3 ubiquitin ligase Pdzrn3 as a regulatory target. We establish that Pdzrn3 is initially phosphorylated shortly after Wnt5a-Ror signaling is activated, a biochemical event which is required for its subsequent proteasomal degradation. From these findings, we develop a flow cytometry-based reporter to monitor Wnt5a-Ror signaling activity in live cells, one of the first of its kind reporters within the non-canonical Wnt signaling field. Using this reporter assay, we delineate the signaling cascade regulating Pdzrn3 phosphorylation and total protein abundance and determine that the C-terminal LNX3H domain of Pdzrn3 is phosphorylated in response to Wnt5a-Ror signaling. A closely related homolog of Pdzrn3, Lnx4, which possesses its own LNXH3 domain, can similarly undergo Wnt5a-Ror-induced degradation, strongly suggesting that the LNXH3 domain is a Wnt5a-responsive domain. We determine that phospho-regulation of Pdzrn3 is necessary for Wnt5a-Ror-driven cell migration. We observed that cell migration is reduced with Wnt5a-Ror signaling activity, suggesting this pathway uses Pdzrn3 abundance to modulate this cell biological process.
In Chapter 3, I question the individual and collective contributions of Dvls to Wnt5a-Ror signaling as well as analyze how RS DVL variants affect signaling in this pathway and Wnt/β-catenin signaling. I develop a dual reporter system derived from mouse embryonic maxillary prominences to measure both Wnt5a-Ror and Wnt/β-catenin signaling in live cells and use a gene knockout and replacement approach in these cells to dissect the function of both wild-type and RS DVL variants. Through this approach, I show that Dvls are required for Wnt5a-Ror signal transduction and functionally non-redundant in dual reporter cells. I also demonstrate that the function of the Dvl C-terminus is to auto-regulate the remaining Dvl domains, which are differentially required in Wnt5a-Ror and Wnt/β-catenin signaling. I identified key residues within the Dvl C-terminus that are phosphorylated during Wnt5a-Ror signaling that are differentially required for Wnt5a-Ror signaling activity but not Wnt/β-catenin signaling, providing insight into Dvl pathway specification. Further, evaluation of RS DVL variant activity in multiple Wnt pathways for the first time indicates that they alter Wnt5a-Ror and Wnt/β-catenin signaling through multiple unique mechanisms, collectively demonstrating that misregulation of these pathways through over- or under-activation contributes to RS. Additional and related work concerning the identification and function of RS-like DVL2 variant present in bulldogs and related dog breeds is included in the Appendix.
Collectively, this dissertation establishes fundamental knowledge regarding Wnt5a-Ror signaling mechanisms in multiple contexts and establishes several practical platforms from which even more detailed interrogation of the pathway can be conducted.
