Yukiko Yamashita didn’t set out to study immortality.
For nearly a decade, her team has studied how sperm-producing stem cells in fruit fly testes divide. But those experiments took the scientists in an unexpected direction. Now, they’ve reported something researchers have never seen: how a crucial snippet of DNA passes in perpetuity to future generations.
This DNA contains genes for the cell’s protein-building machinery, the ribosome. But it also has a fatal flaw. During cell division, some of these genes accidentally pop out of the genome and disappear. If flies lost too much of this ribosomal DNA, or rDNA, every generation, the result would be disastrous. Their capacity to build proteins would steadily drop and the species would be doomed to extinction. “Obviously, that’s not happening,” says Yamashita, a Howard Hughes Medical Investigator at the Whitehead Institute for Biomedical Research.
How these cells, called germline stem cells, retain their rDNA and continue to create copies of themselves indefinitely – how they stay immortal – had eluded scientists for decades. In summer 2021, Yamashita’s team had a breakthrough. Using a technique to visualize rDNA in fly testes, they showed for the first time that stem cells use a built-in mechanism to hang on to these essential genes. The team reported their work in two papers first posted on bioRxiv.org and later published in Science Advances (July 2022) and PNAS (May 2023).
“This is a huge paradigm shift,” says R. Scott Hawley, a geneticist at the Stowers Institute for Medical Research who was not involved in the study. Scientists previously thought that the recovery of rDNA genes from generation to generation was up to chance, though no one had observed the phenomenon directly.
Yamashita’s team instead showed in detailed microscopy images that stem cells can drive the process. Her work, Hawley says, resolves a longstanding conundrum underlying how the cells maintain their rDNA genes. “At a certain point, you actually need to see the things,” he adds. “And that’s what Yukiko has been able to do.”
A fresh perspective
Fruit flies that don’t have enough rDNA can look a little weird. They have difficulty making protein, which leads to appearance oddities – unusually short, thin bristles on their backs, for example, or uneven stripes.
These homely flies, however, can have normal-looking offspring. Somehow, the next generation recovers lost copies of rDNA, keeping the flies’ protein-making machinery intact. Scientists discovered this phenomenon over 50 years ago, dubbing it “rDNA magnification.”
At the time, and for years afterward, the details underlying rDNA magnification were fiercely contested. Scientists in the field largely fell into two rival camps, with differing views on how new rDNA made it into fly chromosomes. “They didn’t agree on anything,” Hawley says.
In 1986, experiments from his groupexternal link, opens in a new tab revealed that the opposing viewpoints stemmed from the groups’ different experimental approaches. At the time, the field lacked the scientific tools needed to probe the question further. Progress on understanding how rDNA genes endured from generation to generation essentially stalled. “I really feel that for 30 years, that was the paper that ended the field,” Hawley says.
Yamashita’s work brought a fresh perspective to the question – though, at first, not intentionally.
Bumping into rDNA
Yamashita’s team uses fruit fly testes to study how germline stem cells divide to produce both a daughter stem cell and a sperm-producing cell. In 2013, they discovered an unexpected quirk in the process. Before a stem cell divides, it duplicates its chromosomes. But copies of the sex chromosomes aren’t randomly assigned to the two resulting cells. One particular copy, the team found, usually ended up in the daughter stem cell.
They investigated whether this chromosome copy had anything unusual in its genome that could explain why it stuck with the stem cell. The researchers identified an unexpected culprit: rDNA. “We were not really trying to study rDNA magnification at all,” Yamashita says. “But we bumped into ribosomal DNA.”
That finding piqued their interest in the mysterious repetitive genes. Yamashita picked up where Hawley and others had left off decades earlier. If daughter stem cells inherit chromosomes with more rDNA copies, her team realized, that could explain how they make up for any rDNA that’s accidentally lost. Based on this idea, the team used a fluorescence microscopy method to visualize rDNA in dividing stem cells by attaching glowing tags to rDNA. The researchers hoped to see more rDNA genes in daughter stem cells compared to daughter sperm-producing cells. But the team’s experiments failed repeatedly. “We were very disappointed,” Yamashita says. “We thought, maybe this is beyond the detection limit.”
Success finally came when postdoc George Watase suggested a different tack: examine those odd-looking fruit flies with fewer rDNA repeats than normal. That would make it easier to spot rDNA magnification, he reasoned, like looking for a needle in a pin cushion rather than a haystack. It worked. Watase caught sight of the rDNA and saw what the team suspected: the chromosome copy with more rDNA genes tended to end up in the daughter stem cell.
Later experiments uncovered more about the underlying mechanisms. During germline stem cell division, a skewed swap of genomic sequences between two identical chromosomes can leave one with extra rDNA. The cells then shunt that chromosome – and its bonus rDNA – to their daughter stem cells. That’s critical for ensuring future generations of flies, Yamashita says. So long as these stem cells have enough rDNA, they can continue manufacturing healthy sperm.
Yamashita’s team’s experiments brought new clarity to the process, revealing something never before observed. “It’s beautiful,” Hawley says.
Yamashita and Watase identified a gene required for the process and named it indra, after a Hindu god who lost immortality. They also showed that retrotransposons are required. Scientists thought that these genes hitchhiked in host genomes without providing useful functions. But flies with low levels of the retrotransposon R2 had trouble producing offspring. Yamashita and her colleagues hypothesize that R2 facilitates the swap that adds extra rDNA to a select chromosome copy.
The team is now diving deeper into how these factors work together to maintain rDNA repeats, a feature present in animals from people to mollusks. “That’s the only way we can start understanding what’s happening in humans,” Yamashita says. “The molecule might not be conserved, but the process will be.”
George J. Watase, Jonathan O. Nelson, Yukiko M. Yamashita. “Non-random sister chromatid segregation mediates ribosomal DNA copy number maintenance in Drosophila.” Science Advances. Published online July 27, 2022. doi: 10.1126/sciadv.abo4443
Preprint posted December 21, 2021, on bioRxiv.orgexternal link, opens in a new tab.
Jonathan O. Nelson et al. “The retrotransposon R2 maintains Drosophila ribosomal DNA repeats.” PNAS. Published online May 30, 2023. doi: 10.1073/pnas.222161312
Preprint posted July 12, 2021, on bioRxiv.orgexternal link, opens in a new tab.