Lab frog DNA shows what happens when genomes collide

The genome sequence of the most common laboratory frog reveals that it originated when different frog species hybridized and the genome doubled, paralleling events that led to the origin of vertebrates.

The genome of the African clawed frog Xenopus laevis (top) is roughly double that of the related western clawed frog X. tropicalis (bottom). (Photo by Atsushi Suzuki, Hiroshima University)

In the Oct. 20 issue of the journal Nature, an international research consortium led by scientists from UC Berkeley and the University of Tokyo reports a striking pattern of genome duplication in the African clawed frog Xenopus laevis. The team showed that the frog’s genome arose through interspecific hybridizations of two now-extinct species between 15 and 20 million years ago.

The newly reported frog genome provides insights into the behavior of anciently duplicated genomes. Several thousand genes have become disabled since the hybridization, and the team found nearly a thousand non-functional “dead” genes, others dying, and still others that had been completely lost. Similar events are thought to have occurred nearly half a billion years ago in the evolutionary lineage leading to modern vertebrates, and the frog provides a contemporary laboratory for studying the impact of genome doubling.

Animals typically have paired sets of chromosomes, with one set inherited from mom and the other from dad. African clawed frogs, however, have twice as much DNA as other frogs, and four sets of chromosomes, a condition known as tetraploidy, Tetraploidy is common in plants and some amphibians, reptiles and fish, but rare in other animals.

By sequencing and analyzing the African clawed frog genome, the researchers found that its four sets of chromosomes were organized as two paired sets, one “long” and the other “short.” (Each parent contributes one long and one short set to its offspring.) Confirming a longstanding hypothesis, the analysis shows that the long and short sub-genomes of X. laevis were originally derived from two conventional frog species. Through a process of interspecies mating, these two progenitors merged to form a new species that eventually became today’s African clawed frog.

Once brought together, the two conventional sub-genomes combined to form a “supergenome” with extra copies of every gene. The two sub-genomes needed to collaborate to guide the functions of the new species, and deal with any conflict between them. Remarkably, the researchers found that the two progenitor genomes responded differently to the merger. While one remained more or less intact, the other shrunk by large- and small-scale deletion and disruption of its genetic material. The result is the “long” and “short” sub-genomes we see today.

The tetraploid African clawed frog in an aquarium, looking at its own reflection. (Photo by Sofia Medina-Ruiz, UC Berkeley)

The international collaboration included researchers from the United States, Japan, Korea, the Netherlands, Australia and Switzerland, and was led by Daniel Rokhsar and Richard Harland of UC Berkeley and Masanori Taira of the University of Tokyo.

The lead authors of the study are postdoctoral fellow Adam Session of UC Berkeley and the Department of Energy Joint Genome Institute, Yoshinobu Uno of Nagoya University and Taejoon Kwon of the University of Texas and the Ulsan National Institute of Science and Technology in Korea.

Two genomes are better than one?

African clawed frogs of the genus Xenopus (zen’-uh-pus, literally “strange foot”) comprise more than 20 species native to sub-Saharan Africa. In the early 20th century, biologists discovered that female frogs respond to human hormones, and that injecting frogs with a woman’s urine could be used for a simple pregnancy test. The frogs became a standard diagnostic tool, and commonplace in hospitals and research laboratories.

Scientists soon realized that Xenopus was also a valuable and versatile laboratory model for basic biology, enabling studies that shed light on both fundamental mechanisms of vertebrate embryonic development and mechanisms underlying human diseases.

“Because X. laevis is such a well-studied model system for cell and developmental biology, it is ideal for exploring the effect of polyploidy on genome evolution,” said Harland, a professor of molecular and cell biology.

Since the 1970s it has been known that many Xenopus species have two, four or even six times as much DNA as most other frogs, along with comparable multiplications of the fundamental chromosome set. In fact, only one conventional diploid species of Xenopus is known.

Although both of the progenitor species are now extinct, the team identified telltale genomic signals that two species were involved, and inferred that they came together 15-20 million years ago to form the species we now know as Xenopus laevis.

“It’s as if the genome we see today was written long ago by two different authors, who each had their own favorite words that they used over and over again,” said Rokhsar. “We used these idiosyncratic words to differentiate the two sub-genomes, figuring out which chromosomes were written by which ‘author.’ We could then analyze the response of these two texts to being merged into a single document.”

The Xenopus laevis genome encodes a frog whose cells and body size are larger than those with smaller genomes. In the millions of years since the merger, there have been subtle, and not-so-subtle, changes in the genome as it adjusted to its new circumstances. In addition to gene loss, the timing of activity during embryonic development has been tweaked for thousands of genes.

“We have only scratched the surface in analyzing the Xenopus laevis genome,” said Taira, a professor of biological sciences at the University of Tokyo. “It is a treasure trove of information for biologists interested in the evolution of genomes and the control of cell and body size.”

The more genomes, the merrier

Duplicated genomes are common in plants, and in some amphibians, reptiles and fish. Scientists believe that mammals cannot survive with duplicated genomes, due to an imbalance in gene function from having too many X or Y chromosomes. But sex determination in frog is not as straightforward as in mammals, and appears to be more flexible, often modulated by environmental factors. This additional flexibility, also found in some fish, seems to have allowed genome doubling to occur in these species.

X. laevis flanked by its smaller cousin, X. tropicalis. (Photo by Atsushi Suzuki)

Genome doublings are more than an amphibian curiosity. Thousands of human genes (and genes of other vertebrates) are found in multiple copies on different chromosomes. These anciently duplicated genes arose nearly half a billion years ago, around the time that early vertebrates began to diverge from other animals. The chromosomal distribution of these duplicate genes in the human and other vertebrate genomes suggests that they are the remnants of ancient genome doublings in an early vertebrate ancestor.

But while hundreds of millions of years have elapsed since these formative genome-doubling events in the evolutionary history of vertebrates, the doublings in Xenopus are far more recent — a mere blink of evolution’s eye, Session said.

“The Xenopus duplication allows us to study a relatively fresh genome duplication, looking at the relatively short-term evolutionary response to doubling,” explained Session. “The ancient vertebrate duplications are far older, and obscured by chromosomal rearrangement and gene loss. In fact, most of the duplicate genes created by the early vertebrate duplications were rapidly lost, because they were redundant. In the frog we can study the ongoing process of loss.”

While many “housekeeping” genes have been lost, duplicate genes that control cell fates tend to be retained, suggesting that their precise dosage is important.

A versatile frog

Xenopus has characteristics that make it an ideal laboratory model, such as large, abundant eggs that can be induced by hormone injection and the ability to lay eggs at any time of year, a result of their sub-Saharan origin, where they must be ready to spawn in response to irregular rains. Xenopus research has led to major advances in understanding cell and developmental biology, and several Nobel prizes.

The tadpole of X. laevis, left, is much larger than the tadpoles of its cousin, X. tropicalis, which has a genome half the size. (Photo by Atsushi Suzuki)

They’re also easy to study, since their development from fertilized frog egg to swimming tadpole takes place in the water. And amphibian embryos are large and and robust enough to allow fine “cut and paste” experiments to ask what parts of the embryo are centers of signaling, and what parts respond to those signals.

In the 1920s, Hilde Mangold and Hans Spemann discovered that a small piece of one embryo, transplanted to another, could induce a second body axis that includes an organized nervous system. In the 1980s, John Gerhart and his colleagues at UC Berkeley found the origins of dorsal-ventral (back to belly) organization of the egg in a rotation of the contents of the egg relative to its surface, a movement that breaks the spherical symmetry. The genes that mediate this play similar roles in human development. Many of these genes were discovered in the 1990s, the first at UC Berkeley by William Smith and Richard Harland. In humans, mutations in these genes can cause birth defects, and Xenopus is an outstanding model for studying these genetic disorders.

Remarkably, cytoplasm extracted from Xenopus eggs and placed in a test tube can carry out most of the events that occur within dividing cells. By partitioning these extracts into their component molecules and then mixing and matching them, researchers have studied many fundamental processes in vertebrate cells. UC Berkeley’s Rebecca Heald uses these extracts to understand the proteins that control assembly and function of the mitotic spindle, which is involved in preparing the chromosomes before a cell divides.

The Xenopus laevis genome is the first amphibian to have its genome sequence reported as complete chromosomes. To achieve this feat, the research team took advantage of new chromatin-related sequencing technologies as well as detailed microscopic examination of chromosomes with fluorescent markers.

At least for now, Xenopus laevis holds the record for the sequenced animal genome with the most genes, with nearly 50,000 genes. But this record is not likely to last long. Along with other diverse frog species, the UC Berkeley team is currently sequencing the genome of a dodecaploid Xenopus species with 12 chromosome sets, expected to nearly triple the record.

The work on the draft genome sequence and genome-wide analysis of Xenopus laevis, conducted by 74 scientists representing 46 institutions, was supported by the U.S. National Institutes of Health and the Japanese and Dutch governments.

RELATED INFORMATION

• Genome evolution in the allotetraploid frog Xenopus laevis (Nature)