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Archive for the ‘Synthetic Vitamin A/ Reinoic acid’ Category

Once upon a time I called myself an artist. I’ve learned much through books. I’ve learned the importance of looking far beneath the surface to understand life and the world I live in. There’s so much more beauty in complexity than only examining skin and it provides far more understanding.

An excerpt from “Your Inner Fish” by Neil Shubin – 2008

HANDY GENES

We begin with an apparent puzzle. Our body is made up of hundreds of different kinds of cells. This cellular diversity gives our tissues and organs their distinct shapes and functions. The cells that make our bones, nerves, guts, and so on look and behave entirely differently. Despite these differences, there is a deep similarity among every cell inside our bodies: all of them contain exactly the same DNA. If DNA contains the information to build our bodies, tissues, and organs, how is it that cells as different as those found in muscle, nerve, and bone contain the same DNA?

The answer lies in understanding what pieces of DNA (the genes) are actually turned on in every cell. A skin cell is different from a neuron because different genes are active in each cell. When a gene is turned on, it makes a protein that can affect what the cell looks like and how it behaves. Therefore, to understand what makes a cell in the eye different from a cell in the bones of the hand, we need to know about the genetic switches that control the activity of genes in each cell and tissue.

Here’s an important fact: these genetic switches help to assemble us. At conception, we start as a single cell that contains all the DNA needed to build our body. The plan for that entire body unfolds via instructions contained in this single microscopic cell. To go from this generalized egg cell to a complete human, with trillions of specialized cells organized in just the right way, whole batteries of genes need to be turned on and off at just the right stages of development. Like a concerto composed of individual notes played by many instruments, our bodies are a composition of individual genes turning on and off inside each cell during our development…

When we compare the ensemble of genes active in the development of a fish fin to those active in the development of a human hand, we can catalogue the genetic differences between fins and limbs. This kind of comparison gives us some likely culprits–the genetic switches that may have changed during the origin of limbs. We can then study what these genes are doing in the embryo and how they might have changed. We can even do experiments in which we manipulate the genes to see how bodies actually change in response to different conditions or stimuli…..We will begin by looking at the structure of our limbs, and zoom all the way down to the tissues, cells, and genes that make it.

MAKING HANDS

Our limbs exist in three dimensions: they have a top and a bottom, a pinky side and a thumb side, a base and a tip. The bones at the tips, in our fingers, are different from the bones at our shoulder. Likewise, our hands are different from one side to the other. Our pinkies are shaped differently from one side to the other. The Holy Grail of our developmental research is to understand what genes differentiate the various bones of our limb, and what controls development in these three dimensions. What DNA actually makes a pinky different from a thumb? What makes our fingers distinct from our arm bones? If we understand the genes that control such patterns, we will be privy to the recipe that builds us.

All the genetic switches that make our fingers, arm bones, and toes do their thing during the third to eighth week after conception. Limbs begin their development as tiny buds that extend from our embryonic bodies. The buds grow over two weeks, until the tip forms a paddle. Inside this paddle are millions of cells which will ultimately give rise to the skeleton, nerves, and muscles that we’ll have for the rest of our lives.

To study how this pattern emerges, we need to look at embryos and sometimes interfere with their development to assess what happens when things go wrong. Moreover, we need to look at mutants and at their internal structures and genes, often by making whole mutant populations through careful breeding. Obviously, we cannot study humans in these ways. The challenge for the pioneers in this field was to find the animals that could be useful windows into our own development. The first experimental embryologists interested in limbs in the 1930s and 1940s faced several problems. They needed an organism in which the limbs were accessible for observation and experiment. The embryo had to be relatively large, so that they could perform surgical procedures on it. Importantly, the embryo had to grow in a protected place, in a container that sheltered it from jostling and other environmental disturbances. Also, and critically, the embryos had to be abundant and available year-round. the obvious solution to this scientific need is at your local grocery store: chicken eggs.

In the 1950s and 1960s a number of biologists, including Edgar Zwilling and John Saunders, did extraordinary creative experiments on chicken eggs to understand how the pattern of the skeleton forms. This was an era of slice and dice. Embryos were cut up and various tissues moved about to see what effect this had on development. The approach involved very careful microsurgery, manipulating patches of tissue no more than a millimeter think. In that way, by moving tissues about in the developing limb, Saunders and Zwilling uncovered some of the key mechanisms that build limbs as different as bird wings, whale flippers, and human hands.

They discovered that two little patches of tissue essentially control the development of the pattern of bones inside limbs. A strip of tissue at the extreme end of the limb bud is essential for all limb development. Remove it, and development stops. Remove it early, and we are left with only an upper arm, or a piece of an arm. Remove it slightly later, and we end up with an upper arm and a forearm. Remove it even later, and the arm is almost complete, except that the digits are short and deformed.

Another experiment, initially done by Mary Gasseling in John Saunder’s laboratory, led to a powerful new line of research. Take a little patch of tissue from what will become the pinky side of a limb bud, early in development, and transplant it on the opposite side, just under where the first finger will form. Let the chick develop and form a wing. The result surprised nearly everybody. The wing developed normally except that it also had a full duplicate set of digits. Even more remarkable was the pattern of the digits: the new fingers were mirror images of the normal set. Obviously, something inside that patch of tissue, some molecule or gene, was able to direct the development of the pattern of the fingers. This result spawned a blizzard of new experiments, and we learned that this effect can be mimicked by a variety of other means. For example, take a chicken embryo and dab a little vitamin A (retinoic acid) on its limb bud, or simply inject vitamin A into the egg. and let the embryo develop. If you supply the vitamin A at the right concentration and at the right stage, you’ll get the same mirror-image duplication that Gasseling, Saunders, and Zwilling got from the grafting experiments. This patch of tissue was named the zone of polarizing activity (ZPA). Essentially, the ZPA is a patch of tissue that causes the pinky side to be different from the thumb side. Obviously chicks do not have a pinky and a thumb. The terminology we use is to the number of digits, with our pinky corresponding to digit five of other animals and our thumb corresponding to digit one.

The ZPA drew interest because it appeared, in some way, to control the formation of fingers and toes. But how? Some people believed that the cells in the ZPA made a molecule that then spread across the limb to instruct cells to make different fingers. The key proposal was that it was the concentration of this named molecule that was the important factor. In areas close to the ZPA, where there is a high concentration of this molecule, cells would respond by making a pinky. In the opposite side of the developing hand, farther from the ZPA so that the molecule was more diffused, the cells would respond by making a thumb. Cells in the middle would each respond according to the concentration of this molecule to make the second, third, and fourth fingers.

This concentration-dependent idea could be tested. In 1979, Denis Summerbell placed an extremely small piece of foil between the ZPA patch and the rest of the limb. The idea was to use this barrier to prevent any kind of molecule from diffusing from the ZPA to the other side. Summerbell studied what happened to the cells on each side of the barrier. Cells on the opposite side often did not form digits; if they did, the digits were badly malformed. The conclusion was obvious. Something was emanating from the ZPA that controlled how the digits formed and what they looked like. To identify that something, researchers needed to look at DNA.

THE DNA RECIPE

That project was left to a new generation of scientists. Not until the 1990s, when new molecular techniques became available, was the genetic control for the ZPA’s operation unraveled.

A major breakthrough happened in 1993, when Cliff Tabin’s laboratory at Harvard started hunting for the genes that control the ZPA. Their prey was the molecular mechanisms that gave the ZPA its ability to make our pinky different from our thumb. By this time his group started to work in the early 1990s, a number of experiments like the ones I’ve described had led us to believe that some sort of molecule caused the whole thing. This was a grand theory, but nobody knew what this molecule was. People would propose one molecule after another, only to find that none was up to the job. Finally, the Tabin lab came up with a novel notion, and one very relevant to the theme of this book. Look to flies for the answer.

Genetic experiments in the 1980s had revealed the wonderful pattern of gene activity that sculpts the body of a fly from a single-celled egg. The body of a fruit fly is organized from front to back, with the head at the front and the wings at the back. Whole batteries of genes are turned on and off during fly development, and this pattern of gene activity serves to demarcate the different regions of the fly.

Tabin didn’t know at the time, but two other laboratories–those of Andy MacMahon and Phil Ingham–had already come up with the same general idea independently. What emerged was a remarkably successful collaboration among three different lab groups. One of the fly genes caught the attention of Tabin, McMahon, and Ingham. They noted that this gene made one end of a body segment look different from the other. Fly geneticists named it hedgehog. Doesn’t the function of hedgehog in the fly body–to make one region different from another–sound like what ZPA does in making the pinky different from the thumb? That parallel was not lost on the three labs. So off they went, looking for a hedgehog gene in creatures like chickens, mice, and fish.

Because the lab groups knew the structure of the fly’s hedgehog gene, that had a search image to help them single out the gene in chickens. Each gene sequence; using a number of molecular tools, the researchers could scan a chicken’s DNA for the hedgehog sequence. After a lot of trial and error, they found a chicken hedgehog gene.

Just as paleontologists get to name a new species, geneticists get to name new genes. The fly geneticists who discovered hedgehog had named it that because the flies with a mutation in the gene had bristles that reminded them of a little hedgehog. Tabin, McMahon, and Ingham named the chicken version of the gene Sonic hedgehog, after the Sega Genesis video game.

Now came the fun question: What does Sonic hedgehog actually do in the limb? The Tabin group attached a dye to a molecule that would stick to the gene, enabling them to visualize where the gene is active in the limb. To their great surprise, they found that only cells in a tiny patch of the limb had gene activity: the ZPA.

So the next steps were obvious. The patterns of activity in the Sonic hedgehog gene could mimic those of the ZPA tissue itself. Recall when you treat the limb with retinoic acid, a form of vitamin A, you get a ZPA active on the opposite side. Guess what happens when you treat a limb with retinoic acid, then map where Sonic hedgehog is active? Sonic hedgehog is active on both sides–pinky and thumb–just as the ZPA does when it is treated with retinoic acid.

Knowing the structure of the chicken Sonic hedgehog gave other researchers the tools to look for it in everything else that has fingers, from frogs to humans. Every limbed animal has the Sonic hedgehog gene. And in every single animal we have studied, Sonic hedgehog is active in ZPA tissue. If Sonic hedgehog hadn’t turned on properly during the eight week of your own development, then you either would have extra fingers or your pinky and thumb would look alike. Occasionally, when things go wrong with Sonic hedgehog, the hand ends up looking like a broad paddle with as many as twelve fingers that all look alike.

We now know that Sonic hedgehog is one of dozens of genes that act to sculpt our limbs from shoulder to fingertip by turning on and off at the right time. Remarkably, work in chickens, frogs, and mice was telling us the same thing. The DNA recipe to build upper arms, forearms, wrists, and digits is virtually identical in every creature that has limbs.

How far back can we trace Sonic hedgehog and other bits of DNA that build limbs? Is this stuff active in building the skeleton of fish fins? Or are hands genetically completely different from fish fins? We saw an inner fish in the anatomy of our arms and hands. What about the DNA that builds it? ….”

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