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Helping the developing brain chart its course

New study in fish shows vitamin A derivative orchestrates timing as 'brain map' forms
A conceptual representation of time controlling neuron development is represented by bright green neurons reaching toward their targets, wearing wrist watches. Only some target areas contain the attracting molecules, shown as red dots.
Zebrafish use time to match motor neurons to the proper throat area during embryonic development. Image courtesy of Ben Hamilton / Developmental Cell

Our brains carry shadowy imprints of our bodies within them, ghostly collections of neurons whose organization reflects the body areas to which they connect. Scientists think that these imprints, known as topographic maps, make it more efficient for our brains to process incoming sensory information and coordinate outgoing motor commands.

These maps form early in our development. But how do they form? How do we ensure that a nerve that helps us speak finds its way to the throat instead of, say, a finger? Working with zebrafish, Dr. Cecilia Moens, a developmental biologist at Fred Hutchinson Cancer Research Center, recently discovered that time is key in shaping the topographic map that represents the muscles of the throat.

Now, in follow-up work published April 16 in the journal Developmental Cell, Moens and her team reveal the molecular cue that controls when the molecular beacons that guide neurons appear. The scientists found that a vitamin A derivative called retinoic acid orchestrates the appearance of these beacons on both sides: in the brain region where the neurons’ cell bodies are, and in the body region into which their long axons stretch.

That coordinated regulation “was completely unexpected to us,” Moens said.

A deeper understanding of how the body develops and maintains this map could help scientists working to repair a dysfunctional or damaged vagus nerve, which can affect speech and swallowing. And, Moens noted, because cancer cells often resurrect early developmental pathways to sustain their growth and spread, the work could give important insights to cancer researchers.

Woman in a blue sweater and white collared shirt.
Dr. Cecilia Moens studies how the brain builds the maps it uses to represent the body. Image courtesy of Cecilia Moens

Topographic maps help improve neural circuit efficiency

Moens has long studied how the nervous system forms its patterns during development.

“Our interest is in the organizing principles of development and a topographic map is an example of a spatially organized group of neurons,” she said. “A topographic map is the way that information from the outside world is represented inside our nervous system.”

Topographic maps appear to help the nervous system generate appropriate responses to incoming sensory information, she said, and improve a neural circuit’s efficiency. Sensory neurons connect from the body to a spatial representation of the body in the brain, and corresponding motor neurons connect from that representation out to the same body areas so incoming information is translated into appropriate outgoing activation through spatial information inside the nervous system, Moens said.

A relatively simple example is a motor reflex in which sensory neurons connected to specific muscles in our arms target motor neurons in the spinal cord that control those same muscles, Moens said. Though there’s a lot of higher order processing that makes our movements smooth and skillful, she noted, the matching spatial organization allows us to generate roughly appropriate reflexive arm movements.

Vagus: the most responsible nerve

Moens and her team study the development of an outgoing topographic map: the motor neurons that carry the brain’s response to sensory input from the throat and the viscera. These are the nerves that control speech and swallowing. In particular, her team tracks the motor neurons that make up the vagus nerve.

This bundle of neurons is the largest nerve of the head and neck and has, Moens said, the most responsibility. “Vagus” means “wandering” in Latin, and the vagus nerve earns its moniker. It carries both sensory and motor information and innervates the throat muscles as well as internal organs. The vagus sends information to the brain about the state of the body, from blood pressure to oxygen and nutrient levels, and it carries information from the brain that regulates everything from breathing rate to bile secretions.

It also controls several important reflexes, including dropping the heart rate in response to rising panic at the sight of blood. (Someone who faints at the sight of blood has an overactive vagal response that drops their heart rate too low.)

Moens’ lab traces the vagus as it projects neurons into the complex segmented structures in the developing embryo that give rise to the muscles that control speech and swallowing. The vessels that carry blood into the head also derive from these structures, which are called the pharyngeal arches.

A dark image showing the vagus nerve projections glowing green as they reach into the developing structures of the zebrafish throat, which glow purple.
Motor neurons (in green) reach from the brain into the segmented pharyngeal arches (in purple). Image courtesy of the Moens Lab

In zebrafish, Moens’ model organism of choice, the pharyngeal arches’ segmented organization is easy to see. In fish, the pharyngeal arches develop into the muscles that control swallowing, as well as another complex task: food sorting.

Fish have taste buds in their throats, not their mouths, and they use their throats to sample food and decide whether to continue swallowing it or to spit it out. They can also use their dexterous throats to remove edible material like algae from stones or other nonfood items before spitting out the stones.

“Fundamentally, we're interested in development of humans and the human nervous system,” Moens said. “Because these cranial nerves are really evolutionarily ancient, we can study their development in the zebrafish and know that we're learning something about fundamental mechanisms underlying nervous system development in humans as well.”

Retinoic acid controls timing

The vagus motor neurons that reach into the pharyngeal arches start out as a cluster in the brain. They don’t all send their long axons racing for the pharyngeal arches at once. Instead, small subgroups of neurons extend their axons at different times: Those closest to the fish’s head start first, then the next group, then the next, ending with those closest to the tail. The pharyngeal arches receive their neurons in the same sequential fashion: The forward-most arch is innervated first, and on down the line as the arches get closer to the fish’s tail.

In this study, Moens and Dr. Adam Isabella, the postdoctoral fellow who spearheaded the work, were hunting for the molecular mechanism controlling this timing. To reveal it, Isabella isolated the neurons innervating different pharyngeal arches at different time points and compared the genes they had turned on. He found that the genes related to retinoic acid signaling were the most different between the neurons that grew early or late during topographic map development.

Derived from vitamin A, retinoic acid plays an important role in patterning major brain areas earlier in development, but the role that Isabella revealed in the vagus motor topographic map was new, Moens said.

Isabella used fluorescent molecules to track the growth of neurons and the presence of retinoic acid. He found that retinoic acid levels changed across the neurons over time. Levels were high in all the neurons before topographic map development. Then, like a tide receding, retinoic acid ebbed across the neurons, starting in the front-most. Neurons didn’t begin growing toward the pharyngeal arches until retinoic acid levels faded. When Isabella blocked retinoic acid from dropping, the neurons couldn’t properly reach the arches.

But how was retinoic acid inhibiting neuron growth? To guide neurons to their targets, the body uses chemoattractants, molecular beacons that help cells navigate to new areas of the body. To be guided, a cell must have the right molecule — known as a receptor — that allows it to sense its beacon.

Moens and Isabella found that retinoic acid prevents neurons from producing their beacon-sensing receptor, called Met. Once retinoic acid levels receded, Met levels went up and neurons were able to begin sending their axons toward the pharyngeal arches.

Moens and Isabella had expected that this timing mechanism only came into play on the neuronal side. It seemed reasonable to assume that sequential release of the neurons would be enough to match them to the proper pharyngeal arch, as each of these also forms sequentially.

Unexpectedly, Isabella found that the molecular beacon drawing the neurons, called Hgf, was also regulated by retinoic acid. It only got turned on to attract the neurons as retinoic acid ebbed.

“At the same time that retinoic acid was releasing expression of Met across the motor neurons, it was also releasing expression of Hgf in the pharyngeal arches so that the matching mechanism could happen in lockstep,” Moens said. “It was more satisfying than surprising to see that you could roll out expression of receptor and ligand in such a regulated way over time to generate a topographic map.”

Presumably, she said, the redundancy is a fail-safe to ensure proper topographic map formation even if the embryo is developing in less-than-ideal conditions.

What purpose does the topographic map serve?

How, exactly, retinoic acid keeps Met and Hgf turned off is one of the narrower questions Moens and her team are now asking. Whether retinoic acid comes from the same source in both areas is another open question.

She’s also thinking bigger. The vagus nerve coordinates several different reflexes in response to different sensory stimuli. It somehow ensures that when we see blood, our heart rate drops to prevent a heart attack, rather than triggering a coughing fit. Does the topographic map make this possible?

“We hypothesize that the topographic map that we have described is set up in order to allow for the matching of sensory information coming in on the vagus nerve and motor information going back out,” Moens said.  

She and her team are working to map the topography of the vagus’ sensory component and see how it matches the motor component they’ve already mapped.

“We’re developing behavioral tests to activate the sensory component of the vagus and visualize what the motor responses are and whether they're spatially organized as well,” she said.

They’re also working to understand whether this map can regenerate after damage, which could have important implications for understanding how to repair a dysfunctional vagus nerve, Moens said.

And though at first glance her work may seem a surprising fit in a cancer research center, it’s not out of place, Moens said.

“Even though we are studying neurons [that are no longer dividing], which can be counted on not to turn into tumors, the signaling pathways that we have discovered to control the development of the vagus topographic map are important cancer targets,” she said.

Cancer cells often reactivate early developmental pathways that allow them to grow and move in ways that normal cells can’t. For example, some tumor cells repurpose Met, the receptor that Moens and Isabella discovered that guides motor neurons, to metastasize, or spread, through the body.

“There are even reports that the antagonistic relationship we have discovered between retinoic acid and Met in the context of the zebrafish vagus motor nerve may also be active in tumor cells, suggesting that retinoic acid could work through this pathway (as well as others) to suppress tumor growth and metastasis,” Moens said. “This underlines the value of discovering the cellular and molecular mechanisms underlying normal development.”

The National Institutes of Health and the American Heart Association funded this work.

Sabrina Richards, a staff writer at Fred Hutchinson Cancer Research Center, has written about scientific research and the environment for The Scientist and OnEarth Magazine. She has a Ph.D. in immunology from the University of Washington, an M.A. in journalism and an advanced certificate from the Science, Health and Environmental Reporting Program at New York University. Reach her at srichar2@fredhutch.org.

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Last Modified, April 17, 2020