Mitochondrial function goes beyond just being “the powerhouse of the cell,” it turns out. A new study shows that the organelles help determine what undifferentiated cells in a growing embryo eventually will become.
After fertilization, an embryo begins as a symmetrical ball of cells with no clear head or tail. But as it develops, signaling within the cells helps determine the embryo’s fate. One critical signaling center for defining the head-to-tail axis is the Spemann-Mangold Organizer. For embryonic development to proceed, mitochondria are an important energy source that drives growth. Now, a Yale-led team has shown that mitochondria also help establish the Organizer, which is critical for normal embryonic development.
The study reveals a new underlying biological mechanism of mitochondrial metabolism and has important implications for treating diseases such as cancer. The researchers published their findings in Developmental Cell on September 5.
“Mitochondria aren’t just providing power, but also signals for different regions of the embryo to turn on different cell types,” says Mustafa Khokha, MD, professor of pediatrics (critical care medicine) and co-principal investigator of the study. “That was a big surprise for us—we were not expecting the mitochondria to do that.”
“We hope we may have discovered a basic tenet of embryonic development,” adds Elizabeth Jonas, MD, Harvey and Kate Cushing Professor of Medicine (endocrinology) and co-principal investigator. “This could help us determine what goes wrong in developmental disorders.”
Leigh syndrome offers first clue to mitochondria function
Outside the laboratory, Khokha cares for many children suffering with congenital malformations. The inspiration for his latest study stems from Leigh syndrome. This is a rare genetic mitochondrial disease caused by mutations in a gene known as LRPPRC.
Children with disorders such as Leigh syndrome cannot metabolize the small molecules in the body typically gained from food. This can lead to metabolic acidosis.
Interestingly, children with Leigh syndrome also often suffer structural abnormalities. Some examples include congenital heart disease and limb deformities. “That’s very unique compared to other metabolic diseases,” says Khokha.
Intrigued, the research team, led by first author Alexandra MacColl Garfinkel, PhD, knocked out the LRPPRC gene in western clawed frog (Xenopus tropicalis) embryos. They hoped to better understand how Leigh syndrome may affect embryonic signaling. Because LRPPRC is critical for mitochondrial function, they predicted that if they could eliminate energy production, the embryo would not develop.
Instead, the researchers saw the Organizer in the embryos expand. “We had a change in patterning of the embryo because we altered mitochondrial function,” says Khokha. “Clearly, mitochondria are not just power sources, but also are involved in patterning.”
In a follow-up experiment, the team then studied the embryos in hypoxia chambers. They wanted to see how the elimination of oxygen, which is crucial for mitochondrial energy production, impacted development.
Once again, they observed an increase in the Organizer. “This highlights a link between the mitochondria and the Organizer,” says Khokha. “When we mess with mitochondrial function, the Organizer gets bigger.”
“If you hamper the mitochondria, which basically prevents energy production, the assumption is everything will die. So, this was a really amazing finding,” Jonas says. “How could knocking out the gene and hypoxia actually expand the Organizer?”
Leaky mitochondria cause developmental abnormalities beyond developmental period
Fascinated yet puzzled, Khokha’s team approached Jonas, whose lab specializes in mitochondrial function. The study, says Khokha, would not have been possible without their teamwork and the collaborative nature of Yale. “Frankly, what to do next wasn’t clear,” he says. “But once we found [Jonas], she knew exactly what to do.”
Jonas had been studying Fragile X syndrome and its associated protein, FMRP. This syndrome causes intellectual disability and abnormal morphological features. Jonas’ lab suspected that this disorder may have underlying metabolic abnormalities, and in 2020, they found that it causes “leaky” mitochondria in which the membranes are not sealed as they should be. “Instead of making the mitochondria not work well, it makes the part of the mitochondria that makes amino acids and lipids stronger,” Jonas explains.
Perhaps the abnormal metabolism they found in Fragile X might actually be normal at an earlier developmental period such as in the embryo.
To answer this question, the researchers traveled to Imperial CoLondon. There, Jonas’ colleague Kambiz Alavian, PhD, who is an adjunct associate professor in endocrinology at Yale School of Medicine, had the only apparatus in the world capable of measuring oxygen consumption within single cells—or in this case single embryos. Using this setup, they discovered that the oxygen consumption of the Organizers was high. “The mitochondria weren’t not working—they were working extra hard,” says Jonas.
Jonas hypothesized that a leaky mitochondrial membrane might be creating an uncoupling of oxygen use from energy production that increases the organelles’ workload. Next, her team placed electrodes on the mitochondria to measure current and found a conductance of current across the membrane, which they later found was due to the ATP synthase c-subunit acting as a leak channel. “This is when we realized the leak could be connected to cell fate determination—how a cell becomes a part of the nervous system or brain or gut,” says Jonas. But what was the signal that the leak produced that did this?
Researchers unveil new pathway underlying mitochondrial metabolism
Through further physiological experiments, the uncoupling of energy production from oxygen consumption, the researchers found, had activated a hypoxia-inducible factor known as Hif-1a. And when they injected Hif-1a into the embryos outside of hypoxic conditions, the Organizer still expanded. “This connected our mitochondria hypoxia phenotype to this single molecule called Hif-1a,” says Khokha. “And then we were off to the races.”
Based on these insights, the team found that the leaky channels on the mitochondrial membrane drove the increased oxygen consumption. This, in turn, led to byproducts that activate Hif-1a.
The study led to a discovery of a novel pathway involving mitochondrial metabolism, Hif-1a, and the Organizer formation. The findings shed light on how mitochondrial metabolism establishes the patterning center of the embryo. Furthermore, they show the crucial role of Hif-1a signaling in expanding the Organizer.
Can better understanding of mitochondria help treat cancer?
Further studies of this altered mitochondrial pathway have critical health implications. Cancer cells, for instance, may have similar activated pathways that drive disease progression.
“While we originally thought about the organizer in terms of developmental biology and how we create the head-to-tail axis, it turns out that a lot of signals that are involved in the Organizer are also involved in many different diseases,” says Khokha. “In embryonic development, these pathways change cell fate, but we think that these same pathways may be used by cancer cells to activate their continued growth.”
In future studies, Khokha and Jonas hope to learn more about why excessive oxygen consumption activates Hif-1a. “There are a lot of different questions to ask,” says Khokha. “And if we can figure this out in the embryo, it’ll be relevant in other contexts as well.”