HOW THE BODY TELLS
LEFT FROM RIGHT.
The precise orientation of the internal organs -----
and those of all other animals with a backbone-----
is controlled in part by proteins that are produced
on only one side of an embryo.
By: Juan Carlos Izpisua Belmonte
L ook in the mirror and draw an imaginary line from the top of your head, along the bridge of your nose, and so on down your chest and your abdomen. You will notice that every external anatom-ical structure on one side of the line has a counterpart on the other side. Yet you have only one heart, one liver, one stomach, one pancreas and one spleen, and your colon coils from your right to your left. Even those organs that come in pairs show some asymmetry: the right lung has more lobes than the left, for instance, and some cerebral structures occur on only one side, or hemisphere, of the brain.
Why do our internal organs defy the symmetry of our overall body plan? And how do they get that way? Attempting to answer these questions, scientists have now identified several of the molecules that dictate organ placement, structure and orientation. We are finding that when these factors are absent or are produced in the wrong place, various human disorders can result. By understanding exactly how the factors function, we may learn how to treat or prevent such diseases.
A Place for Everything...
A symmetric organ structure and placement seem to have evolved because they offer advantages for survival. The very complex digestive system of higher vertebrates—organisms with a backbone—can be more efficiently packed in the body cavity, for example, if the system follows an asymmetric pattern of loops and turns. Similarly, an. asymmetric. heart is better able. to pump and. distribute blood.. Such.cardiac asymmetry allows for two separate.pumping systems: one for directing blood to.the lungs, where it can take up oxygen and discharge carbon dioxide, and a second for delivering the re-oxygenated .blood. to the body.. Interestingly, internal organs can develop in.a mirror-image fashion to the usual arrangement and still work properly.. Approximately.one in every 8,000 to 25,000 people is born with a condition known as. situs. inversus, .in.which the positions of all the internal organs.are reversed relative to the normal situation.(situs solitus.): the person’s heart and stomach lie to the right, their liver to the left, and so on. (The organs are also mirror images of their normal structures.) These people are usually healthy, suggesting that as long as all a person’s organs turn and loop with a specific pattern or internal logic, the actual direction of turning and looping is not important.
... And Everything in Its Place
P eople who are born with abnormally placed organs that are not a mirror image of the usual pattern are not so fortunate . Such individuals, who are said to have situs ambiguus, often die at an early age from lung or heart complications. Others who are born with a condition known as isomerism essentially have two left sides or two right sides to their bodies, so that they either have two spleens or no
spleen, for instance. And internally, their hearts are exactly symmetric. The spectrum of disorders experienced by people with isomerism is complex, but for reasons that are still unclear those with left isomerism fare better than those with right isomerism. Many people with left isomerism have no symptoms at all, whereas those with right isomerism rarely survive beyond one year.
Researchers have elucidated some of the mechanisms that control left-right asymmetry by studying the early stages of heart development in embryos. They concen-trate on the heart because it is the organ most sensitive to abnormalities in the biological machinery controlling the body plan.
All asymmetric organisms begin as symmetric embryos. As far as anyone can tell, all vertebrate embryos are perfectly bilaterally symmetric at the earliest stages of development, with the left side an apparently perfect mirror image of the right side. But at some point early on, this evenness is broken . In vertebrates, the first obvious indication is a very specific event during the initial stages of forming the heart.
The heart arises from two symmetric groups of precardiac cells (the so-called heart fields) that fuse as development progresses forming au initially symmetric heart tube.
The first visible asymmetry is the bending of this tube to the right. This “looping” of the heart tube is one of the most crucial steps in heart formation because it determines the internal structure of the two pumping systems.
In 1995 Clifford J. Tabin of Harvard Medical School, Claudio D. Stern of Columbia University and their colleagues identified one of the biochemical factors that induces looping of the developing heart tube . Studying chick embryos, these researchers found that a protein playfully named Sonic hedgehog is required. (This protein got its name because when a version of it is lacking in fruit fly larvae, the maggots appear rounded and bristly, like frightened hedgehogs.) Specifically, they observed that right looping occurs only if Sonic hedgehog is secreted solely on the left side of a clump of embryonic cells known as Hensen’s node. (Hensen’s node is the location where cells in an early chick embryo sink below other cells to create a three-dimensional embryo; a similar node occurs in mammals.) If Sonic hedgehog appears on the right side of the node instead, the developing heart loops to the left in Sonic hedgehog is not the only player determining the left-right asymmetry of the vertebrate heart. Other known proteins include Nodal and Lefty, which are secreted exclusively on the left side of an early embryo, and Activin BB, Snail and Fibroblast Growth Factor-8, which are only on the right side. When the proteins are made in their correct locations at the appropriate times during development, normal organ placement results; if the location or timing of production of any of these proteins is perturbed, abnormalities occur.
In chick embryos, for instance, the presence of Sonic hedgehog and Nodal on the left side of Hensen’s node and Activin BB on the right leads to a normally asymmetric heart. Applying extra Sonic hedgehog or Nodal protein to the right side of an embryo (so that both sides of the node are now exposed to Sonic hedgehog or Nodal) can override the effects of Activin BB and confuse development: approximately half the embryos will have normal heart looping, but the heart tubes of the other half will loop in the opposite direction. The explanation for this random response seems to be that some additional factor or factors induce looping per se; in that casc, Sonic hedgehog, Nodal and Activin BB influence the direction of looping. Production of Sonic hedgehog on both sides of the node leads to production of Nodal on both sides as well. Lacking clear signals as to which way to loop, each embryo “decides” on the direction of curvature randomly, resulting in 50 percent situs solitus and 50 percent situs inversus.
Interestingly, the result is the same when Sonic hedgehog or Nodal is absent from both sides. Thus, the complete absence of signals in the node or the presence of signals on both sides of the node results in random heart looping. These proteins, like all others, are made when the genes that specify their make-up are active, or switched on. It is not yet known whether people with situs inversus or isomerism have mutations in the genes for the human versions of the Sonic hedgehog and Nodal proteins. but researchers speculate that is the case.
What controls the asymmetric placement and shape of other organs? A gene recently identified by six independent research groups, including mine, seems to be part of the answer . It codes for a protein named Pitx2. Like Sonic hedgehog and Nodal, Pitx2 appears on the left side of the nascent heart and influences the direction of looping. But unlike those substances, it continues to be produced asymmetrically late into embryonic development. Moreover, it is made throughout that period on the left side of organs that are asymmetric.
Manipulating the production of Pitx2 by inserting extra copies of its gene into an embryo results in isomerism or in reverse looping of the gut and other organs as well as the heart, probably depending on the levels of the protein being made. These studies, together with experiments in which the Pitx2 gene is inactivated, indicate that Pitx2 is one of the first factors to establish “leftness during embryonic development . But exactly how Pitx2 and other factors result in looping of the heart tube, the coiling of the gut or the asymmetric development of the brain is still unclear.
Another open question relates to how the initial asymmetry of the body is established. What prompts the production of Sonic hedgehog, Activin BB or Lefty in the first place? One possibility is vitamin A. Over the past few years, researchers have discovered that vitamin A affects the types of cells that arise in an embryo as well as an en embryo’s ability to tell left from right, head from tail and back from front. They have also made great progress in understanding how vitamin A exerts these effects.
My group and others have observed, for instance, that an excess of a form of vitamin A called retinoic acid can even out the normal asymmetry of the heart in rodents and birds. It seems to do so by perturbing the production of proteins such as Nodal, Pitx2 and Lefty. Thus, it appears that the establishment of left-right asymmetry requires the exquisite regulation of vitamin A production during the early stages of embryonic development.
Other factors are certainly involved as well. Accumulating evidence suggests that specialized cell structures called cilia play a pivotal part. Cilia are whiplike structures on the outer membrane of specialized cells, such as those that line the gut; they also allow sperm to swim. Scanning electron microscopy studies have shown that all cells in the nodes of mouse embryos display a single motile cilium, located in a central position on the cell surface. The ciliated cells face the ventral (belly) side of the embryo.
In the human condition known as Kartagener’s syndrome, patients have defective cilia in several cell types, including sperm. These people are prone to developing respiratory infections (because they lack the cilia that normally sweep microbes out of the airways), and males are infertile. In addition, the patients tend to have situs inversus . Similarly, mice that carry a mutant form of a protein that is a component of cilia display randomized organ placement. The obvious conclusion is that the absence of functional cilia in the node causes organ positioning to be determined at random.
THE WHIP FACTOR
A stonishing findings are beginning to clarify how cilia in the node help to ensure normal organ placement. In 1998 Nobutaka Hirokawa of the University of Tokyo and his colleagues observed that mouse nodal cells, which extend their cilia into the fluid surrounding the embryo, rotate their cilia counterclockwise, in a uni-directional motion that has never been seen in other cilia. This motion, in turn, creates a flow of fluid that could sweep critical factors such as retinoic acid, Nodal and Lefty to the left side of the node. That accumulation of fluid and proteins on the left may then provide the bias required to break the initial embryonic symmetry. In other words, a feature of cellular architecture (the direction of rotation of cilia in the node) is translated into a left-right bias in embryonic development that effectively controls the way our internal organs develop.
No one understands just why the cilia rotate in a counterclockwise fashion. Presumably, though, that pattern arises because the molecules driving cilia motion are themselves asymmetric . Nevertheless normal asymmetric organ placement occurs in half the individuals (people or mice) that have absolutely no cilia in their nodes. It follows that nodal cilia are not required for organ development. Rather they are needed to establish the molecular gradients that are required for the proper orientation and positioning of the organs.
When cilia are absent, the preferred flow of extraembryonic fluid fails to materialize; consequently, the left or right determinants carried by the fluid appear on both sides of the node . In such cases, organ position is established at random, presumably because of the random predominance of the appropriate chemical signals on one side of the node or the other.
The problem of left-right determination in the developing embryo has fascinated many biologists for decades, but until very recently progress was slow, in part because of the lack of molecular data. The recent discovery of genes that are active asymmetrically in the early embryo has uncovered many new clues. When some of the genes involved in a particular developmental process are known, researchers can turn them on or off in differing parts of an embryo in the laboratory to test hypotheses about the roles played by the proteins those genes encode. Although the exact nature of the initial event that establishes asymmetry in the embryo is still elusive, identification of proteins involved later on should facilitate discovery of proteins involved in other aspects of organ development. This knowledge may lead to the identification of formerly unknown mutations that predispose to specific organ malformations, which, in turn, will help in developing new systems of prenatal diagnosis.
JUAN CARLOS IZPISIDA BELMONTE received his Ph.D. from the University of Valencia and the Colegio de Espafla in Bologna in 1987. He later moved to the European Molecular Biology Laboratory in Heidelberg, Germany, and, more recently, to the University of California, Los Angeles. He is currently an associate professor of developmental bioogy at thc Salk Institute, La Jolla. Calif. for Biological Studies and the University of California, San Diego. Belmonte has made many contributions to the study of the molecular basis of limb development and left-right asymmetry in vertebrates. Before becoming a scient tist, Belnionte played professional soccer in Alicante, Spain. The author wishes to acknowledge the important contributions of his colleagues Concepción Rodriguez-Estehatt and Javier Capdevila.
June 1999. (Pgs. 46-51)
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