Studying the growth of a LIMB

In studying limb development,biologists are learning how the diversity of limbs—from bird wings to whale flippers—evolved. They are also getting clues that may someday make it possible to regenerate tendons or even entire limbs. But for many experts on limb development,their most important discoveries are how the rules for limb-building also apply to other parts of the embryo.

“The lessons learned in the limb give you insights into how you build a face,or how you build a heart,” said Clifford Tabin,a developmental biologist at Harvard Medical School.

It was not until the early 1900s that developmental biologists ran experiments to understand the forces at work in embryo development. Developmental biologists would snip out pieces of embryos or graft parts together and watch how the development of the embryo was altered. Limbs proved to be the easiest part of an embryo to study. “The limb is totally external,it’s easy to work with,and it’s totally expendable,” Tabin said. “No matter what you do to it,the embryo is going to be fine. Having a heart matters a lot to an embryo. But having a limb doesn’t.”

Chickens became a favourite animal for developmental biologists who studied limbs. “You actually break the eggshell and make a window,and you can cover it with tape afterwards,” said Cheryll Tickle of the University of Bath. “The embryo will continue to develop,and you can find out what happens later on.”

In the 1940s,a Johns Hopkins University biologist,John Saunders,discovered through some slicing and dicing that there were two parts of the limb bud that had mysterious powers over the entire limb’s fate. One of those parts was a translucent ridge that formed along the outer edge,where the fingers eventually form. If Saunders clipped off the ridge,the entire limb stopped developing. If he grafted a second ridge onto a limb bud,it grew

into two arms.

That patch of tissue was called the apical ectoderm ridge. Saunders also discovered a zone on the lower edge of the limb bud,around the place where the pinky would later develop. It somehow sent signals across the limb bud,telling the cells where they were along the pinky-to-thumb axis of the hand and thus which digit to become,and it became known as the zone of polarising activity. When Saunders grafted an extra zone to the thumb side of a limb bud,he produced a second set of digits,arranged in a mirror image to the normal ones.

In 1993,Tickle and her colleagues discovered that the ridge produces a growth-stimulating molecule called,descriptively,fibroblast growth factor. A limb bud could still grow without its ridge,they found,if they implanted in it a microscopic bead soaked with this growth factor.

Tabin and his colleagues discovered another signalling chemical of importance in the limb. Geneticists called it sonic hedgehog,after a video game character. “We know most of the genes now,” said Rolf Zeller of the University of Basel in Switzerland. “We’re trying to understand how these different genes work together.”

Limb development researchers have found that the first steps take place while an embryo is still a crumpled tube. Along the length of an embryo’s flanks,a series of segments forms. Each segment produces chemical signals. And at the places where the shoulders and the hips will be,the signals tell the outer cells to grow rapidly and form little pockets,into which other cells stream. As the pocket grows,it forms the necessary ridge,which sends out other signals telling the cells just underneath it to multiply. As the limb bud grows,the ridge moves away from the cells at its base,which receive fewer growth factors. Without that stimulation,the cells grow more slowly and begin to develop into cells that produce cartilage. They form clumps that will eventually turn into limb bones.

Meanwhile,cells near what will become the pinky start making sonic hedgehog. That molecule spreads across about half the limb bud,to where the middle finger will later form. Cells that produce sonic hedgehog are exposed to the protein the longest. Some experiments suggest that being exposed for a long time turns limb bud cells into pinkies. No time exposed to sonic hedgehog turns them into thumbs.

Each part of the limb knows what it should develop into thanks to sets of genes,each laying down the coordinates in one of three dimensions,and all working together. It turns out,for example,that the cells in the ridge can function only if the limb bud can make sonic hedgehog. Zeller and his colleagues have discovered why: sonic hedgehog switches on a gene in nearby cells called gremlin. Gremlin,in turn,inhibits a protein called BMP4 (for bone morphogenetic protein). At high enough levels,BMP4 can shut down the production of the growth factor in the ridge. So by keeping BMP4 levels low,sonic hedgehog lets the ridge continue to function. Once the limb has reached the right proportions,it must quickly stop growing.

Today,researchers still have much left to learn about the development of limbs. “Your knuckle and your humerus are the same size when they first form,” Tabin said. “Why does the humerus grow so much bigger than the knuckle? We don’t know.”
Tabin argues that long before scientists find the complete pattern of limb growth,they will discover many important insights. It is now clear,for example,that genes involved in making limbs are important for building other parts of the body as well. “Within an embryo the same molecules are used over and over again,” Tabin said. “No one would have expected there would have been so few signals used to form an embryo. If you have a signal that says make a heart,you wouldn’t expect it to make a limb. But that’s exactly what you find.”
_CARL ZIMMER, NYT