Pregnancy that lasts long enough to support full fetal development is a hallmark evolutionary breakthrough of placental mammals - a group that includes humans. At the center of this is the fetal-maternal interface: the site in the womb where a baby's placenta meets the mother's uterus, and where two genetically distinct organisms - mother and fetus - are in intimate contact and constant interaction. This interface has to strike a delicate balance: intimate enough to exchange nutrients and signals, but protected enough to prevent the maternal immune system from rejecting the genetically "foreign" fetus.

To uncover the origins and mechanisms behind this intricate structure, the team analyzed single-cell transcriptomes - snapshots of active genes in individual cells - from six mammalian species representing key branches of the mammalian evolutionary tree. These included mice and guinea pigs (rodents), macaques and humans (primates), and two more unusual mammals: the tenrec (an early placental mammal) and the opossum (a marsupial that split off from placental mammals before they evolved complex placentas).

A Cellular "Atlas of Mammal Pregnancy"

By analyzing cells at the fetal-maternal interface, the researchers were able to trace the evolutionary origin and diversification of the key cell types involved. Their focus was on two main players: placenta cells, which originate from the fetus and invade maternal tissue, and uterine stromal cells, which are of maternal origin and respond to this invasion.

Using molecular biology tools, the team identified distinct genetic signatures - patterns of gene activity unique to specific cell types and their specialized functions. Notably, they discovered a genetic signature associated with the invasive behavior of fetal placenta cells that has been conserved in mammals for over 100 million years. This finding challenges the traditional view that invasive placenta cells are unique to humans, and reveals instead that they are a deeply conserved feature of mammalian evolution. During this time, the maternal cells weren't static, either. Placental mammals, but not marsupials, were found to have acquired new forms of hormone production, a pivotal step toward prolonged pregnancies and complex gestation, and a sign that the fetus and the mother could be driving each other's evolution.

Cellular Dialogue: Between Cooperation and Conflict

To better understand how the fetal-maternal interface functions, the study tested two influential theories about the evolution of cellular communication between mother and fetus.

The first, the "Disambiguation Hypothesis," predicts that over evolutionary time, hormonal signals became clearly assigned to either the fetus or the mother - a possible safeguard to ensure clarity and prevent manipulation. The results confirmed this idea: certain signals, including WNT proteins, immune modulators, and steroid hormones, could be clearly traced back to one source tissue.

The second, the "Escalation Hypothesis" (or "genomic Conflict"), suggests an evolutionary arms race between maternal and fetal genes - with, for example, the fetus boosting growth signals while the maternal side tries to dampen them. This pattern was observed in a small number of genes, notably IGF2, which regulates growth. On the whole, evidence pointed to fine-tuned cooperative signaling.

"These findings suggest that evolution may have favored more coordination between mother and fetus than previously assumed," says Daniel J. Stadtmauer, lead author of the study and now a researcher at the Department of Evolutionary Biology, University of Vienna. "The so-called mother-fetus power struggle appears to be limited to specific genetic regions. Rather than asking whether pregnancy as a whole is conflict or cooperation, a more useful question may be: where is the conflict?"

Single-Cell Analysis: A Key to Evolutionary Discovery

The team's discoveries were made possible by combining two powerful tools: single-cell transcriptomics - which captures the activity of genes in individual cells - and evolutionary modeling techniques that help scientists reconstruct how traits might have looked in long-extinct ancestors. By applying these methods to cell types and their gene activity, the researchers could simulate how cells communicate in different species, and even glimpse how this dialogue has evolved over millions of years.

"Our approach opens a new window into the evolution of complex biological systems - from individual cells to entire tissues," says Silvia Basanta, co-first author and researcher at the University of Vienna. The study not only sheds light on how pregnancy evolved, but also offers a new framework for tracking evolutionary innovations at the cellular level - insights that could one day improve how we understand, diagnose, or treat pregnancy-related complications.

The research was conducted in the labs of Mihaela Pavličev at the Department of Evolutionary Biology, University of Vienna, and Günter Wagner at Yale University. Wagner is Professor Emeritus at Yale and a Senior Research Fellow at the University of Vienna. The study was supported by the John Templeton Foundation and the Austrian Science Fund (FWF).

Kovalev, EIPOD Postdoctoral Fellow at EMBL Hamburg's Schneider Group and EMBL-EBI's Bateman Group, is a physicist passionate about solving biological problems. He is particularly hooked by rhodopsins, a group of colorful proteins that enable aquatic microorganisms to harness sunlight for energy.

"In my work, I search for unusual rhodopsins and try to understand what they do," said Kovalev. "Such molecules could have undiscovered functions that we could benefit from."

Some rhodopsins have already been modified to serve as light-operated switches for electrical activity in cells. This technique, called optogenetics, is used by neuroscientists to selectively control neuronal activity during experiments. Rhodopsins with other abilities, such as enzymatic activity, could be used to control chemical reactions with light, for example.

Having studied rhodopsins for years, Kovalev thought he knew them inside out - until he discovered a new, obscure group of rhodopsins that were unlike anything he had seen before.

As it often happens in science, it started serendipitously. While browsing online protein databases, Kovalev spotted an unusual feature common to microbial rhodopsins found exclusively in very cold environments, such as glaciers and high mountains. "That's weird," he thought. After all, rhodopsins are something you typically find in seas and lakes.

These cold-climate rhodopsins were almost identical to each other, even though they evolved thousands of kilometres apart. This couldn't be a coincidence. They must be essential for surviving in the cold, concluded Kovalev, and to acknowledge this, he named them 'cryorhodopsins'.

Rhodopsins out of the blue

Kovalev wanted to know more: what these rhodopsins look like, how they work, and, in particular, what color they are.

Color is the key feature of each rhodopsin. Most are pink-orange - they reflect pink and orange light, and absorb green and blue light, which activates them. Scientists strive to create a palette of different colored rhodopsins, so they could control neuronal activity with more precision. Blue rhodopsins have been especially sought-after because they are activated by red light, which penetrates tissues more deeply and non-invasively.

To Kovalev's amazement, the cryorhodopsins he examined in the lab revealed an unexpected diversity of colors, and, most importantly, some were blue.

The color of each rhodopsin is determined by its molecular structure, which dictates the wavelengths of light it absorbs and reflects. Any changes in this structure can alter the color.

"I can actually tell what's going on with cryorhodopsin simply by looking at its color," laughed Kovalev.

Applying advanced structural biology techniques, he figured out that the secret to the blue color is the same rare structural feature that he originally spotted in the protein databases.

"Now that we understand what makes them blue, we can design synthetic blue rhodopsins tailored to different applications," said Kovalev.

Next, Kovalev's collaborators examined cryorhodopsins in cultured brain cells. When cells expressing cryorhodopsins were exposed to UV light, it induced electric currents inside them. Interestingly, if the researchers illuminated the cells right afterwards with green light, the cells became more excitable, whereas if they used UV/red light instead, it reduced the cells' excitability.

"New optogenetic tools to efficiently switch the cell's electric activity both 'on' and 'off' would be incredibly useful in research, biotechnology and medicine," said Tobias Moser, Group Leader at the University Medical Center Göttingen who participated in the study. "For example, in my group, we develop new optical cochlear implants for patients that can optogenetically restore hearing in patients. Developing the utility of such a multi-purpose rhodopsin for future applications is an important task for the next studies."

"Our cryorhodopsins aren't ready to be used as tools yet, but they're an excellent prototype. They have all the key features that, based on our findings, could be engineered to become more effective for optogenetics," said Kovalev.

Evolution's UV light protector

When exposed to sunlight even on a rainy winter day in Hamburg, cryorhodopsins can sense UV light, as shown using advanced spectroscopy by Kovalev's collaborators from Goethe University Frankfurt led by Josef Wachtveitl. Wachtveitl's team showed that cryorhodopsins are in fact the slowest among all rhodopsins in their response to light. This made the scientists suspect that those cryorhodopsins might act like photosensors letting the microbes 'see' UV light - a property unheard of among other cryorhodopsins.

"Can they really do that?" Kovalev kept asking himself. A typical sensor protein teams up with a messenger molecule that passes information from the cell membrane to the cell's inside.

Kovalev grew more convinced, when together with his collaborators from Alicante, Spain, and his EIPOD co-supervisor, Alex Bateman from EMBL-EBI, they noticed that the cryorhodopsin gene is always accompanied by a gene encoding a tiny protein of unknown function - likely inherited together, and possibly functionally linked.

Kovalev wondered if this might be the missing messenger. Using the AI tool AlphaFold, the team were able to show that five copies of the small protein would form a ring and interact with the cryorhodopsin. According to their predictions, the small protein sits poised against the cryorhodopsin inside the cell. They believe that when cryorhodopsin detects UV light, the small protein could depart to carry this information into the cell.

"It was fascinating to uncover a new mechanism via which the light-sensitive signal from cryorhodopsins could be passed on to other parts of the cell. It is always a thrill to learn what the functions are for uncharacterised proteins. In fact, we find these proteins also in organisms that do not contain cryorhodopsin, perhaps hinting at a much wider range of jobs for these proteins."

Why cryorhodopsins evolved their astonishing dual function - and why only in cold environments - remains a mystery.

"We suspect that cryorhodopsins evolved their unique features not because of the cold, but rather to let microbes sense UV light, which can be harmful to them," said Kovalev. "In cold environments, such as the top of a mountain, bacteria face intense UV radiation. Cryorhodopsins might help them sense it, so they could protect themselves. This hypothesis aligns well with our findings."

"Discovering extraordinary molecules like these wouldn't be possible without scientific expeditions to often remote locations, to study the adaptations of the organisms living there," added Kovalev. "We can learn so much from that!"

Unique approach to unique molecules

To reveal the fascinating biology of cryorhodopsins, Kovalev and his collaborators had to overcome several technical challenges.

One was that cryorhodopsins are nearly identical in structure, and even a slight change in the position of a single atom can result in different properties. Studying molecules at this level of detail requires going beyond standard experimental methods. Kovalev applied a 4D structural biology approach, combining X-ray crystallography at EMBL Hamburg beamline P14 and cryo-electron microscopy (cryo-EM) in the group of Albert Guskov in Groningen, Netherlands, with protein activation by light.

"I actually chose to do my postdoc at EMBL Hamburg, because of the unique beamline setup that made my project possible," said Kovalev. "The whole P14 beamline team worked together to tailor the setup to my experiments - I'm very grateful for their help."

Another challenge was that cryorhodopsins are extremely sensitive to light. For this reason, Kovalev's collaborators had to learn to work with the samples in almost complete darkness.