Neural Crest Specification requires cytoneme-dependent Wnt signalling

Intercellular communication is fundamental to the functionality of all multicellular organisms. This complex process underpins embryonic development, allowing a fertilised egg to differentiate into a whole organism and facilitating adult tissues in their response and adaptation to injury. Cellular signalling mechanisms involve the transmission of molecular signals that orchestrate transcriptional and behavioural changes in recipient cells. Traditional paradigms suggest that signalling ligands are secreted by source cells and diffuse through the extracellular space to bind receptors on target cells. Although such a mechanism can explain some signalling processes, it fails to explain how specific cells in a progenitor population can be targeted or a precise temporal regulation of signalling can be achieved. Emerging evidence has started to change our understanding of dissemination mechanisms, revealing the existence of cytonemes for cell-to-cell communication. These actin-based cellular threads extend from the signalling cell, enabling precise targeting by delivering molecular signals with high specificity and efficiency over multiple cell diameters to the receiving cell. In a further unexpected twist, we find that cytonemes also transfer pre-formed and active ligand-receptor complexes to target cells to uncouple the endogenous presence of receptors and to increase the signal-to-noise ratio. Neural crest (NC) is an essential cell population in vertebrates that gives rise to, for example, skin pigment cells, peripheral nervous system (PNS), and facial bones. The formation of NC in embryogenesis is characterised by the induction and subsequent migration of a multipotent cell population from the dorsal margin of the neural tube. This process is orchestrated by a sophisticated interplay of external cues and transcriptional networks, which guide the NC cells through their multipotent journey, enabling them to adopt all the diverse cellular fates. The external cues provided by the Wnt signalling family play pivotal roles at multiple stages in the specification and differentiation of NC cells. For instance, Wnt signalling is crucial for the induction of NC identity and ensures that these cells respond to local environmental cues, directing their migration and proliferation. Wnt signalling is then crucial for NC cells to differentiate into pigment-producing melanocytes at the expense of PNS neurons. However, our knowledge about the spatio-temporal control of Wnt signalling during NC specification is fragmented. How can specific cells in this highly migratory population be activated, whereas their neighbouring cells are not? In parallel, the expression of appropriate Wnt receptors in these NC cells is lacking, leaving the mystery of how these cells can respond to the signal. Our recent breakthroughs allow us to explain this conundrum. We hypothesise that dorsal neural tube cells form cytonemes that are essential for transporting the Wnt signal to a subset of NC cells. We further propose that these cytonemes transmit pre-formed and active Wnt ligand-receptor complexes to activate the signalling in a short, well-controlled time window in specific NC cells during migration. To test the hypothesis, we will employ high- and super-resolution in vivo imaging to describe the transport of Wnt signalling components from the Wnt-producing cells of the dorsal midbrain to the Wnt-receiving NC progenitors and signal activation herein. We will use state-of-the-art genetics to establish how cytoneme-mediated signalling allows NC cells to adopt precise and reproducible fates in living zebrafish larvae. Overall, the findings of this project will shift our conceptual framework of precise spatial and temporal precision of cell signalling in vivo.