טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBin-Nun Naama
SubjectProtein Tyrosine Kinase 7 (PTK7) Interacts with the
Canonical Wnt Signaling Pathway in the Developing
Xenopus laevis Nervous System
DepartmentDepartment of Medicine
Supervisor Professor Dale Frank
Full Thesis textFull thesis text - English Version


Abstract

During early Xenopus laevis development, maternal Wnt activity induces the Spemman organizer signaling center on the dorsal side of the embryo. The organizer induces the CNS, which is later modulated in a distinct anteroposterior pattern requiring later zygotic Wnt activity. Wnt signaling is crucial for proper embryogenesis and adult tissue homeostasis. The best characterized Wnt signaling pathway is the "canonical" Wnt/β-catenin pathway. Current models describing the Wnt/β-catenin pathway suggest that following Wnt ligand binding at the cell membrane, the LRP6 and Frizzled (Fz) receptors bind Disheveled (Dsh) protein, undergoing co-aggregation, triggering the stabilization and activation of β-catenin as a transcription factor. The Wnt planar cell polarity (PCP) pathway is another Fz and Dsh mediated Wnt pathway, and is considered one of the "non-canonical" Wnt pathway. Wnt-PCP controls cell polarization, and is necessary for cell orientation and morphogenesis. A new Wnt-PCP regulator was recently characterized, the protein tyrosine kinase 7 (PTK7), which is expressed in the developing Xenopus neural plate. PTK7 is a transmembrane protein that regulates vertebrate PCP, controlling neural convergent-extension (CE) cell movements and neural tube closure in vertebrate embryos. Based on the morphologic phenotypes of PTK7 knocked down embryos, PTK7 was claimed to be a positive modulator of Wnt-PCP activity. We challenge this assumption, and using more sophisticated Wnt-PCP assays, we show that PTK7 actually inhibits Wnt-PCP activity. Thus, PTK7 knock down triggers excess PCP activity that perturbs CE. We also show that PTK7 protein knock down inhibits Wnt/b-catenin activity. Canonical Wnt signaling caudalizes the neural plate via direct transcriptional activation of the Meis3 TALE-class homeobox gene. We discovered that PTK7 controls Meis3 gene expression to specify posterior tissue and downstream Wnt-PCP activity. Furthermore, PTK7 morphants phenocopy embryos depleted for Wnt3a, LRP6 and Meis3 proteins. PTK7 protein depletion inhibits embryonic Wnt/b-catenin signaling by strongly reducing LRP6 protein levels. LRP6 protein is known to positively modulate Wnt/b-catenin activity, but negatively modulate Wnt-PCP activity. PTK7 and LRP6 proteins physically interact, suggesting that PTK7 stabilizes LRP6 protein levels to simultaneously regulate both canonical and non-canonical Wnt activities in the embryo. Search for interaction domains between LRP6 for PTK7 proteins was somewhat inconclusive, but by assaying different deletion constructs, we determined that a PTK7-LRP6 interaction site is possibly present in the LRP6 membrane domain. Both LRP6 and PTK7 proteins similarly regulate b-catenin and Wnt-PCP activities, thus suggesting a novel role for PTK7 protein as a required component of the Wnt/b-catenin pathway and a negative regulator of Wnt-PCP activity.