Scientists at the 91̽»¨ have discovered that a common type of cell in the vertebrate immune system plays a unique role in communication between other cells. It turns out that these cells, called macrophages, can transmit messages between non-immune cells.

Their paper, Feb. 16 in the journal , describes how pigment cells in a species of fish have co-opted macrophages to deliver messages important for pigment patterning in skin. This is the first reported instance of macrophages relaying messages over a long distance between non-immune cells. But since the macrophages are common to all vertebrates, the researchers believe their discovery is no quirk of aquatic life. Macrophages may be common interlocutors for long-distance messages among cells.

“If pigment cells have figured out how to use macrophages for signaling, it stands to reason that others have as well,” said senior author and 91̽»¨biology professor . “This could occur in a variety of cells and animals.”

Parichy and lead author , a 91̽»¨postdoctoral researcher, discovered this new role for macrophages while studying zebrafish. They had wanted to understand how the zebrafish gets its of silver-yellow and black. Each color — black, yellow and silver — arises from a different type of pigment cell. When zebrafish are juveniles, these pigment cells migrate to the right spot to create the stripes.

“As they migrate, communication among these three populations of pigment cells is critical to forming the stripes we see in adult zebrafish,” said Parichy.

Eom and Parichy used laboratory genetic tools to make zebrafish pigment cells glow fluorescent colors — making these cells easier to track using a microscope. In the process, they discovered that xanthoblasts — the precursors to yellow pigment cells — produced unique, elaborate projections during the peak time for pigment pattern formation.

“Xanthoblasts sent these thin projections out in circuitous, almost random directions,” said Parichy. “The projections would eventually encounter another pigment cell, the black melanocyte, and stop.”

Eom discovered that these projections — which they named “airinemes” for mathematician and astronomer Sir George Airy, who described the optical limits to view small objects, as well as the Greek messenger goddess Iris — contained tiny, membrane-bound packages of proteins that provide molecular signals to melanocytes, the black pigment cells. The researchers showed that when an airineme from a xanthoblast encountered a melanocyte, the signal proteins from the airineme would cause the black pigment cell to migrate into the stripe.

Video: xanthoblast sends out airinemes, which take circuitous paths.
Credit: Dae Seok Eom/David Parichy/91̽»¨

But they didn’t understand how airinemes found melanocytes, or why they took such a seemingly random route, until Eom made a critical observation.

“I saw a macrophage interacting with an airineme, and then another, and then another,” said Eom. “In one experiment, I counted 178 airinemes coming from xanthoblasts and 94 percent of them were obviously associated with a macrophage.”

Macrophages are constantly on the move. In fish, people and everything in between, they wander the tissues of the body, “crawling” along like amoebae. Along the way, they sample their environment, picking up and ingesting debris. Their scavenged prizes are often harmless cellular detritus. But if they ingest a bit of a pathogen, or receive signals that a cell nearby is under assault from an invader, macrophages can alert other cells of the immune system.

Armed with this knowledge, Eom tested whether macrophages were truly facilitating the dialogue between yellow and black pigment cells. Using genetic tools, he created zebrafish without macrophages and saw that xanthoblasts produced far fewer airinemes. And under these conditions, melanocytes did not migrate properly to form stripes.

Under the microscope, Eom captured images and movies of how macrophages behaved when they randomly encountered an airineme. A macrophage would seemingly “engulf” one of the round, globular protein packages on the airineme and drag it along, stretching the airineme out.

“Now we know why airinemes seem to take such a meandering, random route,” said Eom. “They are being dragged by macrophages that are themselves moving along randomly.”

But when that same macrophage encountered a melanocyte, the macrophage appeared to “hand off” the airineme to the melanocyte and wander away, presumably delivering the message — via the airineme — to the melanocyte.

Video: a macrophage (purple) drags an airineme from a xanthoblast (green) to a melanocyte (magenta). When the airineme stabilizes on the melanocyte (arrow), the macrophage wanders away. The left panel shows only macrophages and xanthophores/airinemes. The right panel shows the same movie, but with melanocytes now visible. Upper left shows elapsed time (hours, minutes). 
Credit: Dae Seok Eom/David Parichy/91̽»¨

Eom showed that airineme membranes contain a type of lipid that is often an “eat me” signal for macrophages, which may explain why the macrophages attach to and drag along these projections. He and Parichy plan to investigate why macrophages do not digest the airinemes and how the airineme is “handed off” specifically to a melanocyte.

But given the macrophage’s tendency to wander and pick up objects, Parichy believes this is unlikely to be the sole instance of macrophage co-option by cells outside of the immune system.

“It’s very plausible that what we’ve seen here occurs in other contexts where macrophages play important roles, from tissue development and regeneration to cancer,” said Parichy. “We can easily see how macrophages might facilitate signaling between cells in a variety of situations.”

The research was funded by the National Institutes of Health.

###

For more information, contact Parichy at dparichy@uw.edu or 206-734-7331 and Eom at dseom@uw.edu or 512-350-9454.

Grant number: NIH R01 GM096906

The findings – the published Aug. 28 in Science and the in the Nov. 6 issue of Nature Communications – give new understanding about genes and cell behaviors that underlie pigment patterns in zebrafish that, in turn, could help unravel the workings of pigment cells in humans and other animals, skin disorders such as melanoma and cell regeneration.

“Using zebrafish as a model, we’re at the point where we have a lot of the basic mechanisms, the basic phenomenology of what’s going on, so we can start to look at some of these other species that have really different patterns and start to understand them,” said , a 91̽»¨professor of and corresponding author on both papers.

Zebrafish, a tropical freshwater fish about 1.5 inches long, belongs to the minnow family and is a popular addition to home aquariums. Adults have long horizontal blue stripes on their sides, hence the reference to “zebra.” These patterns have roles in schooling, mate selection and avoiding predators. Given their importance, scientists have long wanted to know where these pigment cells come from and how they make stripes and other arrangements.

— In the video clip, a 10-day-old zebrafish gets its stripes in this series of images taken one a day for 30 days. Credit required: D Parichy Lab/U of Washington

Unlike humans with a single pigment cell type – the amount of melanin that produces color being determined by everyone’s individual genetics – there are three pigment cells that make the zebrafish pattern.

Researchers at 91̽»¨and elsewhere have previously shown that all three types of pigment cells communicate with one another to organize zebrafish stripes and that two of the pigment cells – one that creates black and another silver – come from stem cells.

In the Aug. 28 issue of Science, two papers report that the cells called xanthophores that produce the color orange don’t come from stem cells as had long been assumed. Instead, they come from pre-existing cells in the embryo. The 91̽»¨researchers also determined the surprising process by which this occurs: cells in the embryo first mature into xanthophores and then, when it’s time to make stripes, these same cells lose their color, increase in number and then turn back into xanthophores with color.

“This is remarkable because cells do not normally lose their mature properties, let alone regain them later,” Parichy said. “Knowing how xanthophores achieve this feat could provide clues to regeneration of tissues and organs without the need for stem cells.”

Even more remarkably, the 91̽»¨authors found that the re-development of orange-producing xanthophores requires thyroid hormone, the same hormone that turns tadpoles into frogs, suggesting that xanthophores undergo their own metamorphosis. At the same time thyroid hormone blocks development of the black cells, setting the proper shade overall.

“In the last 10 to 15 years people trying to understand these patterns have concentrated on how the three pigment cell types interact with each other. We showed the tremendous dependence on thyroid hormone for the pattern that develops,” Parichy said.

Lead author is , a postdoctoral fellow in Parichy’s lab. Funding for the work was provided by the National Institutes of Health, which just awarded Parichy a new $1.25 million grant to study thyroid hormone signaling in pigmentation and melanoma.

Next in the Nature Communications paper, Parichy’s group reports on a gene that drives the unusually early appearance of xanthophores – independent of thyroid hormone – in another species, the pearl danio. Unlike zebrafish this species lacks stripes: its pigment cells are intermingled and arranged uniformly on the body, giving it a pearly orange color.

By expressing this gene the same way in zebrafish, the researchers caused the fish to make extra-early xanthophores and the fish produced a uniform pattern like the pearl danio instead of their usual stripes.

“Really simple changes in timing make totally different patterns,” Parichy said.

This unexpected result shows that a core network of interacting cells can generate very different patterns in response to changes in timing, a discovery that could explain color pattern evolution across a variety of species. Lead author on the Nature Communications paper is postdoctoral scholar and the work was funded by the NIH.

“If you’d asked me five years ago if we’re in a position to have some useful hypotheses about where patterns come from in other species, I’d have said, ‘No,'” Parichy said. “But I think now we’re really at the point where we understand a lot of the basics and we can start to frame testable hypotheses. We can see how much of this is just a simple difference in timing, a difference in thyroid hormone responsiveness or a difference in cellular communication itself.”

Patterson and UW’s Emily Bain are co-authors on both papers. Other co-authors on the Science paper are UW’s Anna McCann, Dae Seok Eom, and undergraduates Zachary Waller and James Hamill, as well as Julie Kuhlman from Iowa State University and Judith Eisen of the University of Oregon.

###

For more information:
Parichy, dparichy@uw.edu, 206-734-7331

Funding
NIH R01 GM096906, NIH R03 HD074787, NIH P01 HD22486, NIH F32 GM090362, NIH K99 GM105874, NIH R01 GM062182