It’s one of the most significant events in Earth’s history: the moment when a sea creature first stepped – or more likely wriggled – onto land. The momentous occasion 400 million years ago opened up a whole new habitat where life on Earth could evolve and spread out, and made that first bold pioneer and its peers the ancestor to everything from dinosaurs to birds to humans. Obviously, scientists would love to know more about what that brave explorer looked like, and have long hunted for their fossils. But genetics offers another way to journey back in time and look at the biology of the first fish to leave the water, and a study published today by University of Chicago scientists suggests that the genetic tools to make those first historic steps were present long before they actually occurred.
In this case, the genetic hunt was inspired by a famous fossil find, the 2004 discovery of Tiktaalik by a team led by University of Chicago evolutionary biologist Neil Shubin. Tiktaalik was described by its discoverers as a “fishapod” – a transitional species between fish and the four-limbed land-dwelling tetrapods. Though Tiktaalik and its cousins had fin-like appendages, the skeletal structure within those fins was more complex than typically seen for an aquatic species, featuring wrist and hand-like compartments that may have allowed it to do “push-ups” and drag itself slowly across land. Such a sophisticated structure probably didn’t develop overnight, leading Shubin to wonder just how far back the genetic program for developing a limb might have existed in fish.
“This is really a case where knowing something about the fossils and the morphology led us to think about genetic experiments,” said Shubin, the Robert R. Bensley Professor of Organismal Biology and Anatomy and senior author of the study in the Proceedings of the National Academy of Sciences. “Tiktaalik and its cousins showed us that this limb compartment is not an utter novelty in tetrapods, as was thought for a long time. So an antecedent of that program must exist.”
To answer that question, Igor Schneider, a postdoctoral research in Shubin’s laboratory, assembled a genetic “time machine” to look at the origin of a genetic switch for limb development. A genetic switch controls the expression of genes – where and when they are turned on during embryonic development. Schneider took the human sequence of a genetic switch called CsB that controls limb development and looked for similar sequences in a diverse group of animals: mouse, chicken, frogs, zebrafish, and the skate. Though very different today, the animals all share a common ancestor some 400 million years in the past, millions of years before Tiktaalik. Looking at what the CsB switches share in common between those distant relatives today offers a glimpse at the biology of their great-great-great-(repeat 100 million times)-grandfather.
Just looking at the sequences revealed many similarities between the CsB switches of fish species and tetrapods. But the real test was to determine whether the switches performed similar functions despite 400 million years of divergent evolution. To test this required a little bit of mad science: swapping gene sequences across species. First, the CsB switch from a mouse was put into a zebrafish embryo, where it was shown to activate gene expression in the distal fin. The reverse experiment – zebrafish CsB into mouse embryo – was even more exciting, as the primitive fish switch successfully activated gene expression in the developing mouse paw (seen at right).
“The genetic switches that drive the expression of genes in the digits of mice are not only present in fish, but the fish sequence can actually activate the expression in mice,” Schneider said. “This tells us how the antecedents of the limb go back in time at every level, from fossils to genes.”
In both experiments, the gene that the switches activated was merely a reporter gene that told researchers where and when the switch was flipped on. The actual genes that cause an appendage to form the skeletal structure for a limb or fin were not present. But could a transplant of the mouse switch and the relevant genes into a fish embryo produce a lab-grown fishapod?
“With the conservation we see over 400 million years of these switches and the things that activate them, it might not be impossible,” Shubin said. “But that’s not something we attempted.”
Instead, now that the researchers have established the similarities of fish and tetrapod CsB switches, they would like to look at the differences. For instance, what genes or gene switches changed in tetrapods to promote the formation of limbs instead of fins? As Shubin’s laboratory demonstrated earlier this year, subtle changes in the timing or location of gene expression can produce very big anatomical changes. Therefore, the first fish to develop limbs capable of pushing its body onto land didn’t need to develop an entirely new developmental program, but only had to tweak the already-existing genetic switch.
“There previously was the idea that these switches had to be generated from scratch de novo, but no, they already existed, they were already there,” said Marcelo Nobrega, assistant professor of human genetics and another author of the study. “Maybe the key was expressing a gene earlier or later or in a specific territory, but it was just a modification of a program that was already encoded in the genomes of fish almost half a billion years ago and remains there to this day.”