A tiny zebrafish has just hatched from its egg. Under a microscope, the slim, translucent fish lies motionless on its side, too young even to swim. The only movement is its beating heart—a pulsating blob of colorless muscle, with just the slightest hint of pink blood pooling at its base.
Kenneth Poss, an HHMI early career scientist at Duke University, bends over the microscope to peer at the heart. At this early stage of life, the single ventricle of the fish’s heart, a contracting chamber that pumps blood to the rest of the body, is a hollow tube composed of only about 120 muscle cells. Within three months, those cells, called cardiomyocytes, will replicate, morph, and spread to form a full-sized adult heart—a dramatic transformation that Poss recently visualized using a colorful new cell-labeling technique.
Poss, a soft-spoken scientist with an easy smile, normally studies adult zebrafish, not embryos or juveniles. For years, he has investigated the zebrafish’s expert ability to regenerate—to repair an amputated fin, injured retina, damaged spinal cord, and more. In 2002, Poss and colleagues demonstrated that zebrafish can fully regenerate their hearts even when as much as 20 percent of the heart muscle is removed. Humans, on the other hand, have a very limited ability to regenerate heart tissue: though scientists have discovered stem cells in the human heart, cardiomyocyte renewal occurs at a rate of only about 1 percent per year, a rate that declines as we age. Thus, after damage, the heart typically forms scar tissue, rather than repairing itself.
|The zebrafish heart is similar to the human heart in many respects. But unlike the human heart, the fish heart closes wounds rapidly and then regenerates tissue to nearly full function. www.BioInteractive.org @HHMI.|
But in 2007, Poss heard about a new technique that temporarily shifted his focus to developing fish, rather than adult fish. Researchers at Harvard University had created a tool—which they gave the whimsical name “Brainbow”—to visualize mouse brain cells by labeling them with a rainbow of colors. Typically, scientists can track only a single cell at a time in a live mouse, fish, or fruit fly. But with the new technique, the Harvard team could follow hundreds of neurons in a live mouse brain simultaneously.
Poss wondered if it might be possible to adapt the technique to track cardiomyocytes in zebrafish. He began by using it to follow how a zebrafish heart develops after fertilization, hoping to uncover clues about how vertebrates, from fish to humans, build complex organs from just a few cells. “Studying how the heart is built during development is a great way to understand how to build it later in life, if you want to, say, reconstruct heart muscle after a heart attack,” he says.
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
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.”