Around 700 million years ago, the animal kingdom began to branch off from single-celled organisms. Now, scientists have uncovered molecular tools that could have assisted the leap – and successfully tested them by creating a mouse from our unicellular ancestor.

For the first time, scientists created mouse stem cells from the genes of a single-celled life form. Stem cells are special because they can make more of themselves and also transform into other cells with different functions. The team used these newly generated stem cells to help form a living, breathing mouse from a developing embryo, according to published findings in Nature Communications.

The discovery was surprising because scientists thought the genes that allowed stem cells to divide and specialize occurred only in animals and certainly not in a single-celled protist group from almost a billion years ago.

“The molecular tool kit of stem cells is much older than we thought previously,” said Ralf Jauch, a study author and stem cell biologist at the University of Hong Kong. “These molecular tools are older than animal stem cells themselves.”

In addition to learning how we evolved to be multicellular, Jauch said, the understanding of this natural evolution can “make better stem cell models” that could help revert disease or even aging.

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The recipe for stem cells in animals

The difference between animals and protists isn’t just the number of cells. Protists, typically unicellular microscopic organisms that aren’t animals, fungi or plants, perform all functions within a cell. But animals are the ultimate delegators: some cells are assigned to one task, while others are in charge of other actions.

“We know that animals, most of them have stem cells because it’s something that you need,” said Alex de Mendoza, a study author at Queen Mary University of London. “You need cells that can divide but at the same time give rise to other cells.”

It was the 2012 Nobel Prize in medicine that helped shed light on what it takes to generate a stem cell in an animal. Stem cell researcher Shinya Yamanaka, who actually made the discovery six years earlier, found that adult cells could be reprogrammed into stem cells by introducing four specific genes: Sox2, Pou5F1, Klf4 and Myc (known as the Yamanaka factors).

Most people assumed those genes were unique to the animal kingdom because a stem cell capability seemed unnecessary in a unicellular organism. About a decade ago, de Mendoza even searched for these genes in protists and similar unicellular organisms during his PhD research. He only had three sequenced genomes to search at the time, but initial analyses didn’t show any of those special stem cell genes.

Then, recent data uncovered a startling revelation. In 2022, de Mendoza and his colleagues searched the genomes again with more available data. In searching about 30 sequences, they found a few that had a version of these Yamanaka factors found in animals – belonging in the “Sox” and “Pou” gene families.

“We found them and we thought, that’s very weird,” de Mendoza said. “We didn’t expect them to be there.”

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A mouse made from genes older than animal life

The genes, the team discovered, were found in a protist about the size of a dust particle called a choanoflagellate – or “collar” flagellate. These protists are the closest living relatives to the animal kingdom, though they may not seem like it at a quick glance. They propel through the water with a whip (or its flagella) like a microscopic tadpole and scoop bacteria into their feeding collar.

Choanoflagellates could offer insight into the evolution and basic cell biology of the first multicellular organism, even as scientists aren’t completely certain what that first multicellular animal looked like.

Choanoflagellates appear similar to a cell type of sponges, which led some scientists to think sponges could have been the first multicellular animal. More recent data, though, suggested it could have been a comb jelly.

In the study, de Mendoza and his colleagues searched sequences of 22 choanoflagellates species and “only found 2 with convincing hits.” The team wanted to test if these newly found Sox and Pou genes from choanoflagellates would perform similar functions as those in animals.

It wasn’t a foregone conclusion that the protist genes would work in the same way. There are around 20 Sox genes in mammals, with a special variation called Sox2 that is important for programming mammalian stem cells. But the choanoflagellate Sox predates all 20 mammalian copies, so it’s unclear the molecular machinery would operate similarly.

Through a series of experiments, Jauch and postdoctoral fellow Ya Gao introduced the genes from the choanoflagellate into mouse cells. Specifically, they replaced a Sox2 gene from a mouse with the similar gene found in the choanoflagellates – successfully reprogramming the cells to stem cell state. To check that it worked, they injected the reprogrammed cells into a developing mouse embryo. The mouse grew to have physical characteristics from its original embryo but also the lab-induced stem cells, which had genetic markers like dark eyes and black fur patches.

“We can swap pieces with critters that we just don’t seem to have anything to do with them. Then suddenly, they can be used to make things that we consider to be very complicated and very essential,” de Mendoza said.

Not all of the Frankenstein-style experimenting worked in the study. The team also introduced the Pou gene found in the choanoflagellate to the mouse cells, but it did not induce stem cells. The issue, de Mendoza explained, was the unicellular Pou gene bound to the DNA in a different way than other animal Pou genes.

The experiments, Jauch said, suggested that Pou needed more evolutionary tinkering before it reached its current function in modern animals.

To take the investigation even further, the team examined what our common ancient ancestor – perhaps of animals and choanoflagellates – looked like. Colleagues at the Max Planck Institute for Terrestrial Microbiology used advanced computer algorithms to trace back through the molecular tree of life, like a molecular time machine to long-extinct ancestors. They found these molecular traits for Sox proteins were present in these ancient sequences, although the team didn’t make a mouse with them. This finding showed the capability is truly ancestral and predated the evolution of animals themselves.

“The findings of this very elegant body of work are very exciting, but not surprising,” said Sandie Degnan, professor of biology at the University of Queensland who was not involved in the study. “I think of unicellular organisms as needing to be a ‘Jack of all trades’ because that one cell has to meet all the needs of staying alive.”

The study agrees and further extends Degnan and her colleagues’ own proposition that the first animal cells were able to transition between multiple states, similar to modern stem cells. She said it logically makes sense that the first animal cells – our last common animal ancestor – had built-in flexibility that could help them cope with challenges from their external environment. This advantage would likely be more favored through natural selection than cells that stayed in a fixed state.

Evolutionary biologist Daniel Richter, who was not involved in the study, said our close animal relatives continue to impress us. This study along with other related recent work shows that we underestimate what our last common ancestor with choanoflagellates was capable of.

The finding “blurs the line between artificial definitions of what it means to be ‘simple’ versus ‘complex,’” said Richter, a researcher at the Institute of Evolutionary Biology in Barcelona.

The team members are still scratching their heads to figure out why a choanoflagellate or our ancient ancestor would have this gene capability. De Mendoza suggested maybe they used it to regulate basic functions, such as cell proliferation, but multicellular animals repurposed it to make complex bodies.

“Evolution doesn’t always need to invent,” de Mendoza said. “Usually, you use whatever you have, and then you build something new from mostly recycled parts.”

(c) Washington Post