"People have been interested in limbs for a long time because they show such variability in different animals," said David Kingsley, PhD, professor of developmental biology, who led the work. "The debate has been how many genes account for these differences."
The study, published in the April 15 issue of Nature, took advantage of a unique species of fish called the threespine stickleback. Pockets of sticklebacks were isolated by geologic changes at the end of the Ice Age 10,000 years ago, with each newly separated population evolving in response to local ecological conditions. A handful of the thousands of populations around the world lost their hind fins and associated spines, probably to avoid local predators that grabbed the fish by those spines.
Kingsley, who is also an associate investigator with the Howard Hughes Medical Institute, said the debate over how limbs evolve has been stymied because most animals that evolved to have fewer or altered limbs also have a host of other genetic changes, making it hard for scientists to tease out the number and location of genetic changes most important for altering the limb. Sticklebacks, with their recent divergence into many distinct populations, present an opportunity to study recent limb evolution.
The group looked at two populations of freshwater threespine sticklebacks that had lost their hind fins. Working with senior co-author Dolph Schulter of the University of British Columbia, the group crossbred a population of Vancouver freshwater sticklebacks with their four-finned marine relatives. All the resulting offspring had hind fins. These four-finned offspring, which had one set of genes from each parent, indicated to the researchers that fish require only a single copy of the saltwater genes to develop hind fins.
The researchers then interbred these finned offspring to produce fish with a range of hind fin lengths. Some fish lacked hind fins entirely while others had the fully formed fins of their saltwater grandparents. Still other fish had partially formed hind fins or structures that were slightly larger on one side.
Graduate student and co-first author Melissa Marks and former postdoctoral scholar Katie Peichel, PhD, found that the presence of hind fins seemed to correlate with changes at one particular location of the fish chromosomes. A few other chromosome regions had smaller effects on the length and shape of pelvic features, but most of the major evolutionary change could be attributed to a single region. Mike Shapiro, PhD, a postdoctoral scholar and co-first author, found that a gene located at that region is the stickleback version of a gene in mice called Pitx1 that, when mutated, causes mice to have greatly reduced hind limbs. These mice often have asymmetric limb and pelvic reductions, much like the sticklebacks.
These experiments helped explain how mutations at Pitx1 could nudge one population of fish toward losing hind limbs, but they don't hint at whether these mutations were a one-time solution or a widely used strategy.
The researchers addressed this larger evolutionary question using a second population of freshwater sticklebacks discovered by co-author Bjarni Jonsson from the Institute of Freshwater Fisheries in Iceland. The Icelandic fish also lack hind fins, but evolved thousand of miles away from the Vancouver fish. Marks bred the two freshwater sticklebacks and produced an aquarium full of fish lacking hind fins.
Breeding the Icelandic sticklebacks to a four-finned saltwater fish once again generated offspring with hind fins. Together, these experiments suggest that the same set of genes was responsible for the loss of hind fins in two widely separated geographic locations.
"It looks like evolution is using this gene repeatedly," Kingsley said.
The work fits into a growing pattern in evolutionary biology. Although animals look dramatically different, in some cases only a few gene changes account for the differences. "It looks like a small number of genes can have large effects," said Shapiro.
The work also addressed how a few gene changes cause large evolutionary shifts. Most genes have many roles in an animal, so mutations kill the animal rather than altering its shape. Mice with mutated Pitx1, for example, die soon after birth. In the sticklebacks, however, the group found alterations in the location of the Pitx1 gene activity. Although the Pitx1 protein doesn't show up in the developing pelvic region of the fish lacking hind fins, the gene still does its normal job in other regions such as in the thymus and olfactory bulbs.
"We think this is how evolution has been able to sidestep major problems. It only subtracts part of what the gene normally does," Kingsley said.
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