Giving Sharks A Hand

Randy Dahn entered my laboratory with a simple but very elegant idea: treat skate embryos just the way Cliff Tabin treated chicken eggs. Randy's goal was to perform all the experiments on skates that chicken biologists had performed on chicken eggs, from Saunders and Zwilling's tissue surgeries all the way to Cliff Tabin's gene experiments. Skates develop in an egg with a kind of shell and a yolk. Skates even have big embryos, just as chickens do. Because of these convenient facts, we could apply to skates many of the genetic and experimental tools people had developed to understand chickens.

What could we learn by comparing the development of a shark fin to that of a chicken leg? Even more relevant, what could we learn about ourselves from all this?

Chickens, as Saunders, Zwilling, and Tabin showed, are a surprisingly good proxy for our own limbs. Everything that was discovered by Saunders and Zwilling's cutting and grafting experiments and by Tabin's DNA work applies to our own limbs as well: we have a ZPA, we have Sonic hedgehog, and both have a great bearing on our well-being. As we saw, a malfunctioning ZPA or a mutation in Sonic hedgehog can cause major malformations in human hands.

Randy wanted to determine how different the apparatus is that builds our hands. How deep is our connection to the rest of life? Is the recipe that builds our hands new, or does it, too, have deep roots in other creatures? If so, how deep?

Sharks and their relatives are the earliest creatures that have fins with a skeleton inside. Ideally, to answer Randy's question, you would want to bring a 400-million-year-old shark fossil into the laboratory, grind it up, and look at its genetic structure. Then you'd try to manipulate its fossil embryos to learn whether Sonic hedgehog is active in the same general place as in our limbs today. This would be a wonderful experiment, but it is impossible. We cannot extract DNA from fossils so old, and, even if we could, we could never find embryos of those fossil animals on which to do experiments.

Living sharks and their relatives are the next best thing. Nobody would ever confuse a shark fin with a human hand: you couldn't ask for two more different kinds of appendages. Not only are sharks and humans very distantly related, but also the skeletal structures of their appendages look nothing alike. Nothing even remotely similar to Owen's one bone-two bones-lotsa blobs-digits pattern is inside a shark's fin. Instead, the bones inside are shaped like rods, long and short, thin and wide. We call them bones even though they are made of cartilage (sharks and skates are known as cartilaginous fish, because their skeletons never turn into hard bone). If you want to assess whether Sonic hedgehog's role in limbs is unique to limbed animals, why not choose a species utterly different in almost every way? In addition, why not choose the species that is the most primitive living fish with any kind of paired appendage, whether fin or limb? Sharks fit both bills perfectly.

Our first problem was a simple one. We needed a reliable source for the embryos of sharks and skates. Sharks proved difficult to obtain with any degree of regularity, but skates, their close relatives, were another matter. So we started with sharks and used skates as our supply of sharks dwindled. We found a supplier who would ship us every month or two a batch of twenty or thirty egg cases with embryos inside. We became a virtual cargo cult as we waited each month for our shipment of precious egg cases.

Work by Tabin's group and others gave Randy important clues to begin his search. Since Tabin's work in 1993, people had found Sonic hedgehog in a number of different species, everything from fish to humans. With the knowledge of the structure of the gene, Randy was able to search all the DNA of the skate and shark for Sonic hedgehog. In a very short time he found it: a shark Sonic hedgehog gene.

The key questions to answer were Where is Sonic hedgehog active?, and, even more important, What is it doing?

The egg cases were put to use as Randy visualized where and when Sonic hedgehog is active in the development of skates. He first studied whether Sonic hedgehog turns on at the same time in skate fin development as it does in chicken limbs. Yes, it does. Then he studied whether it is turned on in the patch of tissue at the back end of the fin, the equivalent of our pinky. Yes again. Now he did his vitamin A experiment. This was the million-dollar moment. If you treat the limb of a chicken or mammal with this compound, you get a patch of tissue that has Sonic hedgehog activity on the opposite side, and this result is coupled with a duplication of the bones. Randy injected the egg, waited a day or so, and then checked whether, as in chickens, the vitamin A caused Sonic hedgehog to turn on in the opposite side of the limb. It did. Now came the long wait. We knew that Sonic hedgehog was behaving the same way in our hands and in skates' and sharks' fins. But what would the effect of all this be on the skeleton? We would have to wait two months for the answer.

The embryos were developing inside an opaque egg case. All we could tell was whether the creature was alive; the inside of the fin was invisible to us.

The end result was a stunning example of similarity among us, sharks, and skates: a mirror-image fin. The dorsal fins duplicated their structures in a wonderful front-to-back pattern, the same kind we saw with experiments in limbs. Limbs duplicate a limb structure. Shark fins duplicate a shark fin structure as do skates. Sonic hedgehog has a similar effect in even the most different kinds of appendage skeletons found on earth today.

One effect of Sonic hedgehog, you may recall, is to make the fingers distinct from one another. As we saw with respect to the ZPA, what kind of digit develops depends on how close the digit is to the source of Sonic hedgehog. A normal adult skate fin contains many skeletal rods, which all look alike. Could we make these rods different from one another, like our digits? Randy took a small bead impregnated with the protein made by Sonic hedgehog and put it in between these identical skeletal rods. The key to his experiment is that he used mouse Sonic hedgehog. So now we have a real contraption: a skate embryo with a bead inside that is gradually leaking mouse Sonic hedgehog protein. Would that mouse protein have any effect on a shark or a skate?

There are two extreme outcomes to an experiment like this. One is that nothing happens. This would mean that skates are so different from mice that Sonic hedgehog protein has no effect. The other extreme outcome would present a stunning example of our inner fish. This outcome would be that the rods develop differently from one another, demonstrating that Sonic hedgehog does something similar in skates and in us. And let's not forget that since Randy is using the protein from a mammal, it means that the genetic recipe would be really, really similar.

Not only did the rods end up looking different from one another, they responded to Sonic hedgehog, much as fingers do, on the basis of how close they were to the Sonic hedgehog bead: the closer rods developed a different shape from the ones farther away. To top matters off, it was the mouse protein that did the job so effectively in the skates.

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