Vertebrate development is regulated by cellular communication by mechanisms of cell fate and cell behavior. These crucial mechanisms are regulated by cellular signaling and in the case of cell fate, cellular signaling results in transcription of developmentally important genes. Communication between cells can also result in regulation of cell behavior by acting on cytoskeletal elements rather than nuclear factors. One of the cellular signals that regulate both cell fate and cell behavior is the family of Wnt signaling molecules. Wnt5a is one of 19 Wnt molecules and has been previously demonstrated to play critical roles in many important processes in embryonic development as well tumor suppression. Despite many studies that lend credence to a pathway that regulates cell behavior for Wnt5a rather than cell fate, the identity of the pathway(s) Wnt5a impinges upon remains unclear. Despite the possibility of Wnt5a signaling through multiple pathways, here, focus is given to the non-canonical Wnt signaling pathway, a pathway that regulates cell behavior, also known as the Wnt/Planar Cell Polarity (PCP) pathway. The involvement of Wnt5a in the Wnt/PCP pathway was demonstrated with a genetic approach: crossing Wnt5a heterozygous mice with mice heterozygous for a component of the Wnt/PCP pathway to uncover genetic interactions in vivo. Hence, Wnt5a X Looptail (Lp) (Wnt/PCP) heterozygous crosses have been performed. Double heterozygotes for this intercross did not exhibit a decrease in viable progeny as compared to the decreased numbers of Lp heterozygotes. These observations demonstrated a lack of genetic interaction between Wnt5a and the PCP pathway. Wnt5a mutants possess phenotypes associated with deficits in the Wnt/PCP pathway, namely convergent extension (CE) defects and neural tube closure defects. However, upon further investigation of the increased penetrance of craniorachischisis in Wnt5a-/-;Lp+/-, Wnt5a mutants do not display the characteristic broadening of the neural floor plate commonly associated with Lp-/-. This supports that Wnt5a and PCP signaling are parallel pathways that have converged to regulate different aspects of CE and neural tube closure. Despite the complexity of Wnt5a and its potential involvement in multiple pathways, dissection of this will explain the broad range of phenotypes observed.

The Wnts, a family of secreted glycoprotein ligands, act through the frizzled (Fz) receptor, a family of seven-transmembrane (7TM) receptor proteins, to mediate intracellular signaling pathways that regulate cell fate determination, cell migration, or both. Whereas many molecular components of the Wnt signal transduction cascade have been identified, it remains unclear how the signal is transduced from the Fz receptors to the cytoplasm. To address this important question, a membrane-based yeast two-hybrid (MbY2H) screen was performed to identify potential Fz-interacting proteins. For this screen, the Frizzled7 (Fz7) protein was used as the bait and a mouse brain library was used the prey. This screen identified resistance to inhibitors of cholinesterase 8 homolog A (Ric8A), a 542–amino acid cytoplasmic protein, along with other proteins as putative Fz7-binding proteins. Ric8A had been studied previously in C. elegans and D. melanogaster for its function in regulating asymmetric cell division as a receptor-independent guanine nucleotide exchange factor (GEF) for Gα proteins. Additional studies in M. musculus and X. laevis further uncovered a role for this protein during gastrulation and neurulation; however, the mechanisms by which Ric8A regulated these processes remained unclear. In this thesis, I show Ric8A to be a bona fide binding partner for both Fz7; that Ric8A can also bind to the phosphoprotein Dishevelled (Dvl); and that both its interaction with Fz7 and Dvl is Wnt-regulated. The spatial and temporal mRNA expression pattern of the Xenopus homologue of Ric8A suggests a potential role in regulating Wnt signaling. The Xenopus homologue of Ric8A was cloned and gain-of-function and loss-of-function approaches in Xenopus uncovered a role for Ric8A in gastrulation and neural tube closure. Additionally, we found inhibition of Ric8A function mechanistically prevents activation of Rac1 which is required downstream of Wnt/Fz signaling during gastrulation. Overall, this study uncovers a novel regulator of Wnt signaling during early development / Biology