Vikas Gupta, a graduate student in Poss’s lab, spent a year designing and testing the technique, which involves expressing red, blue, and yellow fluorescent proteins in different ratios—like mixing different amounts of the primary colors to create a variety of hues—inside zebrafish cells. Soon, he was able to label zebrafish cardiomyocytes with over 20 colors. Because each cell retains its color permanently and passes the color to its offspring as it divides, these colorful cardiomyocytes are relatively easy to track as they replicate and move around the heart.

		The Genesis of a Technicolor Heart View a slideshow of a developing heart labeled with the Brainbow technique.

Gupta and Poss created dozens of zebrafish with vibrantly colored cardiomyocytes and then examined the fish hearts at select moments between hatching and adulthood. Their results, described in the April 26, 2012, issue of Nature, were beautiful—and surprising.

The researchers found that three distinct events occur during heart development. First, a handful of cells from the ventricle wall—which is only a single cell thick—bud off into the inside of the ventricle and form an internal mesh of muscle.

Next, the remaining cells in the wall multiply and expand laterally, stretching outward like the surface of an expanding balloon, while maintaining a single-cell thickness. Poss was surprised to see that this expansion does not occur in a uniform, predictable pattern. Instead, some cells replicate just a few times while others replicate hundreds of times, resulting in muscle patches of various shapes and sizes, resembling multicolored camouflage. What’s more, no two zebrafish hearts have the same final design, suggesting that a founding population of cells builds the heart in many different ways, not by a single, predetermined plan.

In the last stage of heart development, the ventricle wall begins to thicken. Again, Poss was surprised by what the coloring method revealed: the outermost layer of heart muscle does not originate from the ventricle wall but from cells inside the ventricle. One by one, cells from the internal mesh of muscle pop out of the ventricle at different locations and take up residence on the outside of the heart, where they begin to rapidly replicate and spread out like a wave, enveloping the ventricle in a thick layer of muscle. It takes an average of only eight cardiomyocytes from inside the ventricle to generate the entire outer layer of muscle. “They weren’t easy to find,” Poss says, smiling, “but we did see them consistently.”

If scientists could engineer or identify cells in the human heart with the same potential to form large patches of muscle, Poss says, they might be able to stimulate those cells to heal scars or damaged tissue after a heart attack. “The ultimate goal of our research is to find manipulations that can enhance tissue regeneration,” says Poss. “These cells could help us.”

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