A New Way to Think About Evolution, Courtesy of the Little Skate

Adapted from a release provided by the Max Delbrück Center, Berlin.

Genes are not the only drivers of evolution. The iconic fins of skates are caused by changes in the non-coding genome and its three-dimensional structure, a research team reports in Nature.

WOODS HOLE, Mass. -- The little skate’s dance on the ocean floor is graceful: its massive frontal fins undulate as it skims beneath a layer of sand. With its mottled sand-colored camouflage, the animal is easy to miss.

An international team of scientists, including a group collaborating at the Marine Biological Laboratory (MBL), has discovered how the skate evolved these cape-like fins by peering into its DNA. They found that the key to the evolution of skate fins lies not in the coding regions of its genome, but rather in the non-coding regions and the three-dimensional complexes it folds into. These 3D structures are called “topologically associated domains” (TADs).

The team describes in Nature this week that genomic changes that alter TADs can drive evolution. Until recently, genome evolution was mostly focused on studying variation at the DNA sequence level, but not in 3D genomic structures.

“This is a new way of thinking about how genomes evolve,” said Darío Lupiáñez, geneticist at the Max Delbrück Center and one of the lead authors of the study.

Tetsuya Nakamura and José Luis Gómez-Skarmeta working with the little skate in the MBL's Whitman Center in 2018. Credit: Megan Costello

“Although we found that unique gene-expression patterns establish exceptionally wide skates fins a while ago, the underlying regulatory changes in the genome have previously remained unknown, ” said co-author Tetsuya Nakamura, developmental biologist at Rutgers University.

“To a great extent, evolution is the history of changing the regulation of gene expression during development,” said the late José Luis Gómez-Skarmeta of the Andalusian Center for Developmental Biology (CABD) in Seville, senior author on the paper, in 2018.

More than 450 million years ago, the genome of a primitive fish — the ancestor of all vertebrate animals — duplicated twice. The expansion in genetic material drove the rapid evolution of more than 60,000 vertebrates, including humans.

One of our most distant vertebrate relatives is the little skate (Leucoraja erinacea), which belongs to a lineage of cartilaginous fishes that includes sharks and rays. These distant cousins are ideal organisms to learn about the evolution of traits that made us human, such as paired appendages.

One of the few places in the world that collects Leucoraja erinacea and breeds it for research, including for the present study, is the Marine Resources Center at the MBL.

"Skates are cartilaginous fishes called Chondrichthyans. They are considered more similar to ancestral vertebrates,” said Christina Paliou, a developmental biologist at the CABD and one of the first authors. “We can compare the characteristics of skates with other species and determine what is novel and what is ancestral."

An exciting time in evolutionary genomics

In 2017, Gómez-Skarmeta, a founding figure in evolutionary genomics, brought together scientists from around the world to study skate evolution: laboratories with expertise in genome evolution such as the Ferdinand Marlétaz lab at University College London and the Daniel Rokhsar lab at University of California-Berkeley; in skate biology such as the Neil Shubin lab at University of Chicago, where Tetsuya Nakamura was then located; and in 3D gene regulation such as the Juan Tena, Dario Lupiáñez and Gómez-Skarmeta labs, as well as other collaborators.

Gómez-Skarmeta was interested in learning how genomes evolve structurally and functionally to promote the appearance of new traits. The Gómez-Skarmeta, Nakamura and Shubin labs pursued this research collaboratively in the MBL Whitman Center in 2018, using skate embryos and husbandry support provided by the MBL Marine Resources Center, led by the late David Remsen, and with research support from the MBL Embryology course.

It was an exciting time for evolutionary genomics. Genome sequencing technologies had significantly improved and scientists could gain novel insights into how DNA, which stretches a couple of meters end-to-end, is folded into a 0.002-inch-diameter cell nucleus.

Team members at the Marine Biological Laboratory in 2018, including co-authors Tetsuya Nakamura (2nd from left), José Luis Gómez-Skarmeta (3rd from lef), Neil Shubin (4th from left), Elisa Calle-Mustienes (5th from left). Credit: Diana Kenney

“The packaging of DNA in the nucleus is far from random,” said Lupiáñez. The DNA folds into 3D structures called TADs, which contain genes and their regulatory sequences. These 3D structures ensure that the appropriate genes are switched on and off at the right time, in the right cells.

Rafael Acemel, a geneticist at the Max Delbrück Center and one of the first authors, performed experiments using the Hi-C technology, to elucidate the 3D structure of the TADs. But interpreting the results was challenging at first as the scientists needed the complete skate genome as a reference point. “At the time, the reference consisted of thousands of small unordered pieces of DNA sequence, so that did not help,” Acemel said.

To overcome this difficulty, the scientists used long-read sequencing technology, together with Hi-C data, to assemble the pieces of the DNA like a puzzle and assign the unordered sequences to skate chromosomes. With the new reference, assembling the 3D structure of the TADs using Hi-C became trivial.

They compared this improved skate genome with genomes of the closest relatives, sharks, to identify any TADs altered during skate evolution. These altered TADs included genes of the Wnt/PCP pathway, which is important for the development of fins. There was also a skate-specific variation in a non-coding sequence near the Hox genes, which also regulate fin development.

“This specific sequence can activate several Hox genes in the front part of the fins, which does not happen in other fish or four-legged animals,” said Paliou. Subsequently, the scientists performed functional experiments that confirmed these molecular changes helped the skates evolve their unique fins.

Living embryo of a little skate sitting atop its yolk at approximately 10 weeks. Photo: Mary Colasanto and Emily Mis, MBL Embryology Course

TADs drive evolution

Earlier research has shown that changes in TADs can affect the expression of genes and cause diseases in humans. In this study, scientists show a role for TADs in driving evolution that has been previously noted for moles, too.

After the primitive fish ancestor duplicated its genome, many unused and redundant parts were subsequently lost. “It was not only the genes that disappeared, but also the associated regulatory elements and the TADs they are contained in,” Lupiáñez said. “I think it’s an exciting finding as it suggests that the 3D structure of the genome has an influence on its evolution.”

TADs are important for gene regulation -- 40 percent of them are conserved in all vertebrates, Acemel says. “However, 60 percent of TADs have evolved in some way or another. What were the consequences of these changes for species evolution? I think that we are just scratching the surface of this exciting phenomenon,” Acemel said.

This mechanism of evolution constrained by TADs could be prevalent in nature. “We suspect that these mechanisms might explain many other interesting phenotypes that we observe in nature,” Lupiáñez said. “By adding these new layers of gene expression, gene regulation, and 3D chromatin organization, the field of evolutionary genomics is entering into a new era of discovery.”

Citation:

Ferdinand Marlétaz, Elisa de la Calle-Mustienes, Rafael D. Acemel, Christina Paliou et al. (2023): The little skate genome and the evolutionary emergence of wing-like fin appendages. Nature, DOI: 10.1038/s41586-023-05868-1

Further information

Selected Articles:

Hunting Down the Enhancers of Evolution: How Does a Fin Become a Limb? (Marine Biological Laboratory, 2018)

Moles: Intersexual and genetically doped (previous example of TADs drinving evolution) (Max Delbrück Center, 2020)

Influential barriers (Max Delbrück Center, 2022)

Selected Literature:

Nakamura T, Klomp J, Pieretti J, Schneider I, Gehrke AR, Shubin NH (2015) Molecular mechanisms underlying the exceptional adaptations of batoid fins. Proc. Natl. Acad. Sci. U S A, DOI: 10.1073/pnas.1521818112

Turner N, Mikalauskaite D, Barone K, Flaherty K, Senevirathne G, Adachi N, Shubin NH, Nakamura T (2019) The evolutionary origins and diversity of the neuromuscular system of paired appendages in batoids. Proc. Royal Soc. B, DOI: 10.1098/rspb.2019.1571

Chiara Anania, Rafael Acemel (2022) In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation. Nature Genetics. DOI: 10.1038/s41588-022-01117-9.

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.