Your Inner Fish

While Shubin and his team dig up fish fossil bones, Randy Dahn at the research lab at the University of Chicago looks at the embryos of sharks and skates (a smaller cousin of a shark. Dahn is investigating the affects of Vitamin A on limb development in sharks, hoping to explain part of the way that our DNA directs body cells to form a functional body. Dahn’s experiments look to compare the DNA “recipe” for shark fins to the “recipe” for human hands.

Experiments on DNA fill in an important gap that fossil study can’t address, as fossils are rare and cannot be manipulated to change specific variables or answer certain questions. Dahn wants to prove that the genes for fish fins and human hands are virtually identical by manipulating shark embryos to make part of the fin look like a hand.

Though the human body is made up of hundreds of different kinds of cells, every cell in an individual human’s body has the exact same DNA in its center. Different organ cells develop differently because only certain genes are active in each cell. Understanding what switches a gene on or off in a particular cell helps explain what genes are involved in specific body systems. Isolating the genetic differences between the code for a fin and the code for a hand gives Shubin likely places to look for a switch that allowed an animal like Tiktaalik to start making the bones for a hand instead of a fin.

Making Hands. Hands have three dimensions: top to bottom, pinky side to thumb side, and base to tip. Shubin looks for the genes that make a pinky look different from a thumb as a “key” to the genetic recipe that controls hands. In the embryo, limbs develop from the third to eighth week after conception. First, tiny buds extend from the body, then form into little paddles. The tips of these paddles are the millions of cells that will become the limb’s skeleton nerves and muscles. Scientists study limbs that have gone wrong because it is easier to identify genetic mutations that differ from the “normal” DNA recipe.

To study embryonic limbs, scientists need an organism that is big enough to see and manipulate and that has readily available, fairly cheap embryos. In the 1940s and 50s, chicken eggs were the perfect candidate. Edgar Zwilling and John Saunders, two scientists that studied embryos, cut into chicken embryos and surgically removed small patches of tissue in the limbs to see what would happen. They discovered that a small zone of tissue is responsible for the development of the entire limb. Removing it at different times in the embryo’s life stops limb development at different junctures.

Mary Gasseling, a member of John Saunder’s embryo lab, transplanted limb tissue to different places on the limb to see how manipulating the place affects limb development. Taking a small patch of tissue from the “pinky” side of the limb bud and transplanting it to the “thumb” side early in development actually causes the embryo to develop a limb with a full duplicate set of digits, arranged as a mirror image of the normal set. Injecting vitamin A into the chicken egg during development produces the same result. The patch of tissue that controls limb development was named the zone of polarizing activity (ZPA).

The ZPA controls the formation of fingers and toes by controlling the concentration (amount) of a certain molecule in the cells that will become the limb. The cells closest to the ZPA have a high concentration of the unknown molecule and respond by making a pinky finger. The cells farther away from the ZPA have a low concentration of the molecule and respond by making a thumb. The cells in between have varying concentrations of the molecule that correspond to making the second, third, and fourth fingers.

The DNA Recipe. In the 1990s, scientists were better able to look for the molecular mechanisms that the ZPA uses to differentiate fingers. Cliff Tabin, Andy MacMahon, and Phil Ingham decided to look at flies for the answer. Genetic experiments in the 1980s had already mapped out the gene activity that guides fly development from front to back, with different genes active in the front head than the back wings. Tabin, MacMahon, and Ingham identified another gene that controlled the body regions of the fly.

Tabin, MacMahon, and Ingham named the gene that controls differentiating body segments in flies the hedgehog gene, because flies that have a faulty hedgehog gene look like little bristly hedgehogs. In chickens, this gene is called “sonic hedgehog” (named after the video game character). Sonic hedgehog is only active in the ZPA of chicken embryo limbs. After experiments that confirmed that hedgehog does the same limb production in flies, chickens, and mice, Dahn began to look for a sonic hedgehog gene activity in sharks.

Sharks and their smaller cousins, skates, have embryos in eggs that are remarkably similar to chicken eggs, with some adaptations for life in water. Looking for sonic hedgehog activity in skate embryos would prove that this basic recipe for limb development goes far further back than just land animals in the history of life on earth, as the earliest shark fossils are dated to 400 million years ago. Sharks and humans are distantly related, and obviously look very different on the surface. Shark bones are even made out of a different material than human bones. All of these differences make it even more useful to use sharks to see if sonic hedgehog is unique to limbed animals or if it is active in all animals with appendages.

Dahn started by looking for the sonic hedgehog gene in shark embryos. Once he found that sonic hedgehog was indeed present, he started to run through all the experiments that Tabin’s team had done on chicken eggs. In each instance, the shark fin reacted the same way as chicken limbs – to the point of producing a duplicate fin when the shark ZPA was treated with Vitamin A.

Dahn went further to see if the shark ZPA could be influenced with the protein that the sonic hedgehog gene produces in mice. Normally, the rods in a shark’s fin are all the same. When Dahn inserted the mouse sonic hedgehog protein, the rods of the shark fin developed to be different sizes and shapes from each other, just like mouse fingers do.

Dahn’s experiments prove that all appendages, whether fins or limbs, develop the same way from the same basic DNA recipe. Shubin argues that this means the transition from fins to limbs did not involve any new DNA, but simply using the ancient genes for fin-making in new ways. Ultimately, experiments on flies, chickens, mice, and sharks show the similarity between all animals with appendages and give insight to the genes responsible for the development of human limbs.