Painting the vagus topographic map with a gRAdient

From the Moens Lab, Basic Sciences Division

The vagus motor neurons, which control swallowing and speech, originate in the hindbrain and innervate the pharyngeal arches (PAs). The word "vagus" derives from the Latin for "wandering", as the nerves wander from the brain into organs in the neck, chest, and abdomen. But the vagus neurons are no mere vagabonds. On the contrary, these neurons are organized in intricate evolutionarily conserved topological maps wherein the spatial organization of the neuron cell bodies mirrors the spatial organization of their axon targets, which can be located in completely different regions of the body. These maps enable the flow of information from the neurons to their target cells. Yet the molecular mechanisms that guide the organization of these topological maps during development are only partially understood. The Moens lab (Basic Sciences Division) studies the development of the neuronal networks connecting the brain to the muscles of the head and neck using zebrafish as a model organism. In a recent study, Moens and colleagues investigated how the topographic maps of vagus motor neurons are formed. They published their findings in a recent issue of Developmental Cell.

The study revealed a new mechanism whereby a gradient of retinoic acid (RA), a known morphogen, organizes the zebrafish vagus motor topographic map by coordinating chemoattractant signaling between Hgf and its receptor Met in a spatiotemporal manner. Dr. Adam Isabella, a postdoc in the Moens lab, led the study. “When we started this study, there was a model called the chemo-affinity hypothesis, for how topographic maps form, which basically suggested that spatially organized signals guide axons to the correct targets.” Isabella said. However, spatial patterning is unlikely to be the only mechanism in play during topographic map formation. In a previous study, researchers from the Moens lab showed that topographic connectivity in the zebrafish hindbrain is regulated simultaneously by both spatial and temporal factors. As Isabella explained: “The most important thing we've found in studying the vagus nerve is that the spatial component was not the only factor at play, and that regulating the timing of signaling can also be an effective way to help axons discriminate between potential targets. It's also been clear for a long time that, for topographic maps to form, the embryo must coordinate the patterning of two tissues - the neural tissue and the target tissue - which are often quite far apart in the body.”

A schematic shows the temporal matching model of topographic vagus motor targeting.
A schematic shows the temporal matching model of topographic vagus motor targeting. From bioRxiv

In zebrafish and in humans, the vagus motor neurons are arranged such that the anterior neurons innervate anterior PAs, while posterior neurons innervate posterior PAs. Understanding the mechanisms underlying these topographic maps is not trivial. “One of the most challenging parts of studying topographic maps is being able to see the map, by which I mean being able to see which neurons are extending to which targets, so that we can understand how that organization changes in different experimental conditions,” Isabella explained. “We developed some tools to reliably label both individual neurons and specific groups of neurons in live animals, and to precisely measure which tissues they innervated, which allows us to get a really high-resolution view of how the different factors we identified were affecting the structure of the map,” he added. Isabella and colleagues took advantage of their abilities to image zebrafish development in real time and to label and isolate specific neurons. By profiling the transcriptomes of the isolated neurons, they discovered that an RA gradient is required for specific axon targeting of vagus motor neurons. Isabella commented on their approach: “These tools were really helpful in that they allowed us to pull labeled neurons out of the animal and use genomic approaches to ask what were the gene expression differences between groups of vagus neurons that allowed them to make different targeting decisions.” In follow-up experiments, the authors found that increasing RA levels induced anterior neurons to project to more posterior targets, whereas depletion of RA caused posterior neurons to project to anterior targets.

How does RA influence axon target in vagus motor neurons? Cues to the answer came from the hindbrain transcriptome, where RA treatment led to the downregulation of met, a gene encoding a receptor tyrosine kinase. The authors found that met is expressed in the anterior axon nucleus, while its chemoattractive ligand hepatocyte growth factor (hgf) is expressed in anterior PAs. The requirement of both Met and Hgf for vagus innervation of the PAs was confirmed by mutagenesis. Together, the data establish a model whereby RA controls the spatiotemporal dynamics of Hgf/Met signaling to coordinate axon targeting to PAs during development.

Future work will be required to determine how this type of organization promotes nerve function. “Topographic maps are very common, if not ubiquitous, in sensory and motor networks, but it's still somewhat of a mystery why this architecture is so important. For the vagus nerve, we think topography allows this nerve to multitask effectively by compartmentalizing its different functions,” Isabella said. “Now that we have the tools to disrupt the map, we're working on tools to measure how these disruptions affect circuit function and behavior,” he added. Isabella is devoted to understanding how this topographic map is repatterned after injury. He explains: “Nerve damage is a big clinical problem, and regeneration seems to be the most promising solution. I've found that the vagus map can regenerate in fish, but it's clear that it's not controlled by the timing mechanism we identified in the embryo, so it would be illuminating to figure out what new mechanisms are allowing axons to find the correct target tissues in this context.”

Isabella AJ, Barsh GR, Stonick JA, Dubrulle J, Moens CB. (2020). Retinoic Acid Organizes the Zebrafish Vagus Motor Topographic Map via Spatiotemporal Coordination of Hgf/Met Signaling. Developmental Cell. http://doi:10.1016/j.devcel.2020.03.017

UW/Fred Hutch Cancer Consortium member Cecilia Moens contributed to this work.

This study was supported by funding from the National Institutes of Health and the American Heart Association.