Molecular understanding of cytoneme-based Wnt trafficking

Cell-to-cell communication by signaling proteins is essential to orchestrate development and tissue homeostasis in all multicellular organisms. The highly conserved family of Wnt proteins are important guiding cues to control these processes. Fundamental to this complex signaling network are relatively small and defined signaling centers in a given tissue that produce and distribute Wnt proteins. Adjacent, larger groups of cells respond to these spatial and temporal information in a concentration-dependent manner and adjust their transcriptional program. However, a regulated sequence of morphogen activity is required to generate a fine tuned communication network. Therefore, a controlled propagation machinery must ensure accurate signal distribution from the source to the surrounding tissue to initiate the correct developmental path. In this thesis, I consolidated the knowledge of the molecular machinery controlling cytoneme formation in zebrafish development. I expanded this principle to other aspects of Wnt signaling such as cancer growth and tissue homeostasis. Via a screening approach, I identified the receptor tyrosine kinase Ror2 as a promoting factor for cellular protrusions in general and particularly for Wnt8a cytonemes in cultured cells and in vivo. Consistently, I described the novel ligand-receptor pair Wnt8a and Ror2 by measuring the affinity for membrane accumulations and by biophysical imaging applications such as fluorescence correlation spectroscopy. Subsequently, functional interaction and transduction of the Wnt/PCP pathway was demonstrated during zebrafish convergence and extension and during non-canonical reporter activation in Xenopus. Wnt8a and Ror2 are considered to act in mutually repressive pathways, although the autocrine interplay for cytoneme formation to facilitate paracrine Wnt/β-catenin dissemination seems to be conserved. Thus, the model can be applied to other systems: The transcriptional β-catenin level and resulting proliferation of gastric cancer cells can be regulated by Ror2, thereby only disrupting the signal transmitting transport machinery in the source cells. Furthermore, I provided evidence of an ex vivo human stem cell organoid system, where growth and survival require cytoneme-mediated Wnt proteins from isolated myofibroblasts. Remarkably, this setup resembles an innovative approach for stem cell maintenance in the murine intestinal crypt and expands the potential roles of cytonemes in development, tissue homeostasis and diseases