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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.

Signaling filopodia, termed cytonemes, are dynamic actin-based membrane structures that regulate the exchange of signaling molecules and their receptors within tissues. However, how cytoneme formation is regulated remains unclear. Here, we show that Wnt/planar cell polarity (PCP) autocrine signaling controls the emergence of cytonemes, and that cytonemes subsequently control paracrine Wnt/β-catenin signal activation. Upon binding of the Wnt family member Wnt8a, the receptor tyrosine kinase Ror2 becomes activated. Ror2/PCP signaling leads to the induction of cytonemes, which mediate the transport of Wnt8a to neighboring cells. In the Wnt-receiving cells, Wnt8a on cytonemes triggers Wnt/β-catenin-dependent gene transcription and proliferation. We show that cytoneme-based Wnt transport operates in diverse processes, including zebrafish development, murine intestinal crypt and human cancer organoids, demonstrating that Wnt transport by cytonemes and its control via the Ror2 pathway is highly conserved in vertebrates., eLife digest Communication helps the cells that make up tissues and organs to work together as a team. One way that cells share information with each other as tissues grow and develop is by exchanging signaling proteins. These interact with receptors on the surface of other cells; this causes the cell to change how it behaves. The Wnt family of signaling proteins orchestrate organ development. Wnt proteins influence which types of cells develop, how fast they divide, and how and when they move. Relatively few cells, or small groups of cells, in developing tissues produce Wnt proteins, while larger groups nearby respond to the signals. We do not fully understand how Wnt proteins travel between cells, but recent work revealed an unexpected mechanism – cells seem to hand-deliver their messages. Finger-like structures called cytonemes grow out of the cell membrane and carry Wnt proteins to their destination. If the cytonemes do not form properly the target cells do not behave correctly, which can lead to severe tissue malformation. Mattes et al. have now investigated how cytonemes form using a combination of state-of-the-art genetic and high-resolution imaging techniques. In initial experiments involving zebrafish cells that were grown in the laboratory, Mattes et al. found that the Wnt proteins kick start their own transport; before they travel to their destination, they act on the cells that made them. A Wnt protein called Wnt8a activates the receptor Ror2 on the surface of the signal-producing cell. Ror2 then triggers signals inside the cell that begin the assembly of the cytonemes. The more Ror2 is activated, the more cytonemes the cell makes, and the more Wnt signals it can send out. This mechanism operates in various tissues: Ror2 also controls the cytoneme transport process in living zebrafish embryos, the mouse intestine and human stomach tumors. This knowledge will help researchers to develop new ways to control Wnt signaling, which could help to produce new treatments for diseases ranging from cancers (for example in the stomach and bowel) to degenerative diseases such as Alzheimer’s disease.