Somewhere in the nighttime dark of the forests of Southwestern Australia, a small marsupial is foraging in the treetops, seeking a lick of sweet tree sap, when it realizes it’s being hunted by a snake. Cornered, it makes a desperate leap into empty space! Then it flings its limbs out and catches the air with its patagium—a flap of skin that expands between its fore- and hindlegs—letting it glide to safety far out of the snake’s reach. In a paper published April 24, 2024 in the journal Nature, the research team of Princeton scientist Ricardo Mallarino, in collaboration with colleagues at the Baylor College of Medicine, explains the developmental and evolutionary processes that produce this handy survival tactic.

Our escape artist is the sugar glider Petaurus breviceps, but two related marsupial species also have patagia. Although they share a common ancestor, the three gliding marsupial species are all perched on different branches of their family tree; each is more closely related to other, non-gliding marsupial species than they are to one another. This implies that, rather than inheriting the patagia from some now-extinct ancestor who could also glide, they each independently evolved their patagia to enable gliding behavior—a process known as convergent evolution.

For Mallarino, an Assistant Professor in Princeton’s Department of Molecular Biology, this poses compelling questions: how do different species arrive at a common physical structure? Does this happen through the same genes and developmental pathways or has each species evolved different solutions to the same problem? Jorge Moreno, a graduate student working under Mallarino’s supervision and co-first author of this study, was likewise intrigued by these questions.

“We don't quite understand how novel traits and adaptations originate from a molecular and genetic perspective. We wanted to investigate how an evolutionary novelty arises,” said Mallarino.

The team had their work cut out for them: to this point, very little was known about patagium development in any species, and genome information was only available for the sugar glider. Moreno and Mallarino worked in collaboration with Baylor scientists Olga Dudchenko and Erez Aiden Lieberman, co-authors in the study who led the effort to sequence the genomes of the other two gliding marsupials, plus 8 closely related non-gliding marsupials and 4 distantly related marsupials. 

In closely related species, physical differences in body structures are often caused by changes in where and when genes are activated to guide the formation of those structures during embryonic development. Activation of these genes is controlled by nearby regulatory DNA sequences found in the genome. Therefore, the team reasoned, the genetic programs that drive patagia formation should be found where gliders’ genomes harbor large numbers of differences in regulatory elements compared to the genomes of closely related non-glider species. Indeed, the team discovered that gliders’ genomes have many changes in regulatory sequences located near the gene Emx2.

If the Emx2 gene is involved in patagium formation, then it should be present in this tissue when it first develops in the young animal, just after birth while the joey is still in its mother’s pouch. The researchers observed that Emx2 is indeed present in the growing patagium, and furthermore, that blocking Emx2 expression there stunts patagium. Later experiments showed that loss of Emx2 affects the activation of many other genes including Wnt5a, a gene the team had previously shown is critical for patagium development. Thus, the researchers theorize that Emx2 is a master controller for patagium formation.

Is this developmental program found in other mammals or is it only present in gliding marsupials? To explore this question, the researchers examined skin development in mice, which are distantly related to marsupials and do not develop a patagium. Surprisingly, these studies showed that Emx2 is active in a similar anatomic region as in sugar gliders, but only for a very short time. Prolonging Emx2 activation in the mouse caused skin to show similar features as during formation of the sugar glider patagium, but it did not result in the appearance of a patagium. This implies that either Emx2 by itself is not enough to trigger patagium formation, or else that mice lack some of the genetic circuitry required to form one, but it also suggests that Emx2’s ability to regulate skin is conserved across evolution. Differences in Emx2 activation patterns may partly explain why some species have a gliding membrane while others don’t. 

“Understanding the underlying changes that happen at the genomic level to give rise to these convergent traits is important because it can tell us whether evolution is targeting the path of least resistance. You can have the same outcome but different paths to get there,” said Moreno.

Mallarino is currently planning experiments to investigate how Emx2 expression is controlled in gliders and to identify the downstream genetic targets of Emx2 that drive patagium development. 

“Gliding membranes have evolved in many other species, including much more distantly related ones,” said Mallarino. Accordingly, he plans to probe whether similar genetic circuits are employed in more distantly related gliding animals, such as flying squirrels, bats, colugos, and even some geckos.

“We would like to understand whether the molecular processes we uncovered here are also present in those other species or whether their membranes have evolved through completely different molecular mechanisms. This can help us understand the extent to which the same processes are recycled over and over throughout evolution or whether there are many different ways to build the same trait across distantly related species,” he said.

Citation: Jorge A. Moreno, Olga Dudchenko, Charles Y. Feigin, Sarah A. Mereby, Zhuoxin Chen, Raul Ramos, Axel A. Almet, Harsha Sen, Benjamin J. Brack, Matthew R. Johnson, Sha Li, Wei Wang, Jenna M. Gaska, Alexander Ploss, David Weisz, Arina D. Omer, Weijie Yao, Zane Colaric, Parwinder Kaur, Judy St. Leger, Qing Nie, Alexandria Mena, Joseph P. Flanagan, Greta Keller, Thomas Sanger, Bruce Ostrow, Maksim V. Plikus, Evgeny Z. Kvon, Erez Lieberman Aiden, and Ricardo Mallarino. Emx2 underlies the development and evolution of marsupial gliding membranes. Nature. 2024. DOI: 10.1038/s41586-024-07305-3.

Funding: The work described here was supported by the National Institutes of Health (R35GM133758, UM1HG009375, RM1HG011016-01A1, F32 GM139240-01, T32GM007388, R01-AR079150); the Searle Scholars Program; the Sloan Foundation and the Vallee Scholars Program; the Welch Foundation (Q-1866), the US-Israel Binational Science Foundation (2019276); the National Science Foundation (DGE-2039656, NSF DBI-2021795, NSF PHY-2210291); the LEO Foundation (LF-AW-RAM-19-400008, LF-OC-20-000611); and the W.M. Keck Foundation (WMKF-5634988). 

Funders: National Institutes of Health, Searle Scholars Program, Sloan, Vallee Scholars Program, Welch Foundation, US-Israel Binational Science Foundation, National Science Foundation, LEO , W.M. Keck Foundation

Grant Numbers: R35GM133758, UM1HG009375, RM1HG011016-01A1, F32 GM139240-01, T32GM007388, R01-AR079150, Q-1866, 2019276, DGE-2039656, NSF DBI-2021795, NSF PHY-2210291), LF-AW-RAM-19-400008, LF-OC-20-000611, WMKF-5634988