ABOVE: Some turtle species can survive freezing temperatures and prolonged periods without oxygen, unique adaptations that scientists struggle to study using currently available tools. MODIFIED FROM © ISTOCK, blueringmedia, gollykim; DESIGNED BY ERIN LEMIEUX

In the dark, frigid months of winter, painted turtle hatchlings hunker down in shallow, icy nests where they enter a state of suspended animation.1 Their hearts stop beating; their metabolisms slow down; and over half of their bodies’ water turns to ice. With the arrival of spring, the baby turtles begin to thaw and signs of life return.

The painted turtle has a remarkable capacity to survive freezing temperatures and prolonged periods without oxygen, which are unique adaptations that make these reptiles ideal models for studying hypoxia, anoxia, and freeze tolerance. Humans and painted turtles share some of the genes that become active in the reptile during low-oxygen conditions, but differences arise in their regulation.2 A deeper understanding of the genes and molecular mechanisms that drive these states could inform new approaches for treating hypoxic injury following a stroke or heart attack as well as cryopreservation strategies for organ transplantation. However, the limited availability of genomic tools and in vitro models for studying the species limits mechanistic insights.

In a study published in Communications Biology, researchers introduced a method for generating stem cell-derived turtle liver organoids.3 The researchers hope that their new technology will facilitate a deeper exploration of the mechanisms driving the turtle’s unique adaptations.

“The idea of this project was to generate tools that can be adopted by other researchers,” said Nicole Valenzuela, an evolutionary biologist at Iowa State University and coauthor of the study. “They can answer the questions that they have wanted to ask but couldn't.”

Valenzuela has worked with turtles for nearly 30 years, focusing on sex determination.4 Her team had identified a number of genes that might regulate this temperature-dependent biological process, but tools for turtle-specific functional genomics did not exist . “We hit a wall,” said Valenzuela. 

In contrast to short-lived, rapidly-reproducing laboratory animals, such as fruit flies and mice, painted turtles reproduce seasonally and live for 20 to 60 years. “They're not your typical model organism,” said Valenzuela. “Turtles have fascinating biology, but they are difficult to study.”

Human cell lines power biomedical research and several technological and therapeutic advances in science have utilized these tools. “We don’t have that with the turtle,” said Kyle Biggar, a biochemist at Carleton University who was not involved in this study. “This is a major limitation.” Instead, turtle biologists have relied on observational studies and terminal experiments that provide a series of descriptive snapshots of signal transduction pathways or metabolic adaptations that are fixed to a single time point. “Before, we were producing hypotheses, but we couldn't test them,” said Biggar. “Organoid research is really interesting because it gives the field an advanced, modern tool to learn more about low oxygen adaptation and potential freeze tolerance.”

A few years back, Valenzuela was at an event on campus where she met researchers in the College of Veterinary Medicine who were developing intestinal organoids from dogs for drug testing.5 Valenzuela’s fascination with the approach grew into a collaboration with the researchers to test whether they could apply this technology to turtles. “This paper gives the answer that is categorically yes,” said Valenzuela. “We just produced the first turtle organoids in the world.”

Photo of Nicole Valenzuela.
Nicole Valenzuela, an evolutionary biologist at Iowa State University, has studied turtles for more than 30 years and is interested in the genes that regulate sex determination, anoxia, and freeze tolerance.
Christopher Gannon, Iowa State University

Valenzuela and her team focused on generating liver models given the organ’s critical role in orchestrating the painted turtle’s superabilities.6 After collecting liver tissue samples from adult painted turtles, the researchers harvested stem cells and provided them with some scaffolding and a cocktail of factors that stimulate growth. Not only did the cells generate organoids, but the researchers also found that they could freeze and revive the cells indefinitely for a continuous source of experimental material. “Even just having a tractable turtle cell line that you can work with is a huge step forward,” said Biggar. Using the same culture protocol, they created organoids from embryonic and hatchling painted turtles as well as two other species of turtle.

With the organoids in hand, the researchers set out to characterize the mini livers and explore how they stacked up to their tissue of origin. The use of classical histology approaches alongside electron microscopy helped confirm that some of the cell types present in the liver also emerged in their organoids. The mini livers were enriched for epithelial cells, specifically cholangiocytes and hepatocytes. To characterize the molecular makeup of the cells, the researchers used RNA sequencing. Most of the genes identified in the organoids were also expressed in the tissue of origin.

The use of different turtle species and life stages allowed the researchers to explore species- and age-specific differences that lay the groundwork for future within- and cross-species experiments. Preliminary transcriptomic and proteomic analyses produced a collection of differentially expressed genes and proteins, respectively, that the researchers can probe further to isolate the molecules that regulate the painted turtle’s unique adaptations.

“The authors did a good job of showing what the potential for these resources are, [but] they didn’t go into actually showing if these organoids are able to adequately respond to periods of low oxygen, which is obviously the next thing to do,” said Biggar. He also noted that oxygen adaptation is a whole organism response that integrates hormonal and environmental signals and involves an interplay between multiple tissues, so the organoid may function differently than the original organ. Despite this limitation, Biggar added, “You certainly still gain a lot of novel insights.” 

Now, turtle biologists have access to a platform that, when used alongside functional genomic tools such as CRISPR gene editing, could facilitate mechanistic experiments. Valenzuela and her team are still optimizing the organoids, but she is excited to test the role of specific genes in regulating freeze and anoxia tolerance. She said the next steps involve tracking the molecular changes that occur when they expose the organoids to different environmental conditions. 

“There’s lots of descriptive work still to be done, but no matter what, it’s an important iterative step towards [larger models of understanding],” said Biggar. 

References

  1. Storey KB, Storey JM. Molecular physiology of freeze tolerance in vertebrates. Physiol Rev. 2017;97(2):623-665.
  2. Shaffer HB, et al. The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage. Genome Biol. 2013;14(3):R28.
  3. Zdyrski C, et al. Establishment and characterization of turtle liver organoids provides a potential model to decode their unique adaptations. Commun Biol. 2024;7(1):218.
  4. Bista B, Valenzuela N. Turtle insights into the evolution of the reptilian karyotype and the genomic architecture of sex determination. Genes. 2020;11(4):416.
  5. Chandra L, et al. Derivation of adult canine intestinal organoids for translational research in gastroenterology. BMC Biol. 2019;17(1):33.
  6. Dinkelacker SA, et al. Anoxia tolerance and freeze tolerance in hatchling turtles. J Comp Physiol B. 2005;175(3):209-217.