Mutants
Page 10
What induces a limb-bud to grow out into space? In 1948 a young American biologist, John Saunders, gave an answer to this question. He had noticed that limb-buds were crowned by a ridge of unusual cells. The cells were clearly ectoderm – the tissue that covers the entire embryo – but at the tips of limbs they resembled tightly packed columns, quite unlike their usual pancake shape. Saunders dubbed this structure the ‘apical ectodermal ridge’ and then, curious to know more, decided to remove it.
As embryonic newts haeve been used to study the organiser, so chickens have been used to study limbs. Saunders operated on twenty-two foetal chickens, some young, others a little older. In each case he removed the apical ectodermal ridge from one wing-bud, while leaving the one of the other side intact. Having operated, he sealed up the egg and waited until the chicks hatched out. The operated wings all had a characteristic deformity: they were, to varying degrees, amputated. Chickens operated on when the limb-bud had just begun to expand showed severe amputations: they had at best a humerus (the bone closest to the shoulderblade), but below that, the radius, ulna, wrist bones and digits were all gone. Those operated on a little later had a humerus, radius and ulna, but lacked wrists and digits; later yet, only the digits were missing.
This experiment helps to explain why some infants, such as Hermann Unthan, are born without arms or legs. Our limb-buds also have apical ectodermal ridges, and sometimes they must surely fail. The ridges on Hermann’s arm-buds probably malfunctioned soon after they first appeared; perhaps they never appeared at all. Other human deformities resemble the less extreme amputations seen in chicks whose wing ridges are removed only late in their growth. In the Brazilian states of Minas Gerais, São Paulo and Bahia there are families who are afflicted with a disorder called acheiropody – from the Greek: a – absence, cheiros – hand, podos – foot. Instead of hands and feet, the victims of this disorder have limbs that terminate in a tapered stump. They get about by walking on their knees and are called by the locals aleijadinhos, or ‘little cripples’. The disorder is caused by a recessive mutation, probably quite an old one since it appears in more than twenty families, all of Portuguese descent. Because the mutation is recessive, only foetuses who have two copies of the mutant gene fail to develop hands and feet. Having two copies of a mutation is usually a sign of inbreeding: the first family of aleijadinhos ever studied were the children of a Peramá couple who were – local opinion varied – either full siblings, half siblings, or else uncle and niece.
ACHEIROPODY. AN ALEL]ADINHO, BRAZIL 19705.
The apical ectodermal ridge is the sculptor of the limb. As the development of the limb-bud draws to a close, the ridge regresses, leaving behind an outline of our fingers and toes. Should it be damaged in any way, the consequences will be visible in the limb’s final form. The ectrodactylous hands of the Wigtown cleppies were the result of a mutation that caused a gap in the middle of the ridge, and so a gap in the middle of the forming limb. Mutations in at least four different genes are known to cause ectrodactyly, but it is quite possible that more will be discovered.
What gives the ridge, which is little more than a clump of cells, such power over the shape of a limb? The most obvious explanation would be that the cells making up the tissues of the limb – bone, sinew, blood vessels and so on – have their origin in the ridge. But this is not the case. All of these tissues are made of the mesoderm that lies beneath the ridge rather than the ridge itself; only the skin is ectoderm. The obvious alternative is that the ridge matters not as a source of cells, but rather as a source of information: it tells mesoderm what to do.
Action at a distance in the embryo usually implies the work of signals, and so it is in the limb-bud. Apical ectodermal ridges are rich in signalling molecules, especially so in one family of them: the fibroblast growth factors or FGFs. The experiment that identified FGFs as the source of the ridge’s power began with the surgical extirpation, à la Saunders, of the apical ectodermal ridge from the tip of a young wing-bud. The denuded bud was not, however, allowed to grow up into the usual amputee wing. Instead, a silicone bead soaked in FGF was placed on its tip, more or less where the ridge would be. The result was a fully-grown limb – one cured, if you will, by the application of a single protein. Twenty-two genes in the human genome encode FGFs, of which at least four are switched on in the ridge. No one knows why so many are needed there, but collectively they are vital to the workings of the ridge. It would be an exaggeration to say that to grow a leg or an arm one needs only a little FGF, but clearly a little goes a long way.
Ridge FGFs not only keep mesodermal cells proliferating, they also keep them alive. Many cells will, at the slightest provocation, commit suicide. They have a whole molecular machinery to assist them in doing away with themselves. Seen through a microscope, a cell suicide is spectacular. Over the course of an hour or so the doomed cell becomes opaque, then suddenly shrivels and disappears as it is consumed by surrounding cells. In the limb-bud, FGFs block the machinery of death; they give cells a reason to live. Yet while mass cell suicide is clearly a bad thing, at least some cell death is needed to form our fingers and toes, for if the ridge is the sculptor of the limb, cell death is the chisel. At day 37 after conception our extremities are as webbed as the feet of a duck. Over the next few days the cells in the webs die (as they do not in ducks) so that our digits may live free. Should a foetus have too much FGF signalling in its limbs, cells that should die don’t. Such a foetus, or rather the child it becomes, has fingers and toes bound together so that the hand or foot looks as if it is wearing a mitten made of skin.
When Saunders removed the apical ectodermal ridge from a young limb-bud, the result was total amputation. Yet if the bud was older and larger, then only the structures further down – wrists, digits – were lost. Why? Over the last fifty years, various answers have been given to this question. The latest, though surely not the last, turns on two quite new observations. The first of these is that the ridge FGFs only penetrate a short way, about two hundred microns (one fifth of a millimetre) into the mesoderm. In a young limb-bud, two hundred microns-worth of seceding cells cuts very deep as a proportion of total mass; in an older, larger limb-bud, much less so. This difference in proportion matters because limb-buds possess an invisible order. A limb-bud may look like an amorphous sack of cells, but even when newly formed, when it is no more than a bump on the foetal flank, its mesodermal cells have some foreknowledge of their fates. Some are already destined to become a humerus, others digits, yet others the parts between. As the limb-bud grows, each of these populations of cells proliferates and expands in turn. When a young limb-bud is deprived of FGFs, all of these variously fated cell populations suffer; when an older limb-bud is deprived only those closest to the tip do, and with them future hands and feet, toes and fingers.
This account of the making of our limbs contains within it the roots of twentieth-century medicine’s most infamous blunder. In 1961 an Australian physician, William McBride, reported a sudden surge in the numbers of infants born with deformed limbs. Similar findings were reported a few months later by a German named Lenz. Both physicians suggested that the defects were caused by a sedative used to prevent morning sickness that has the chemical name phtalimido-glutarimide, but which swiftly became notorious by its trade-name, thalidomide. More reports rolled in from around the world. By the time it was all over, more than ten thousand infants in forty-six countries with thalidomide-induced teratologies had been found. Only the United States escaped the epidemic because a few sceptical FDA officials had delayed authorisation of a drug that was, at the time, the third best-selling in Europe.
The thalidomide infants had a very particular kind of limb deformity. Unlike acheiropods, their limbs did not suggest amputations in the womb, for most had reasonably formed hands and feet as well as shoulderblades and pelvises; they were simply missing everything else in between. Without long bones, their arms and feet connected almost directly to their torsos. Their limbs had the appearance of flip
pers – a condition dubbed phocomelia or ‘seal-limb’.
Phocomelic infants have always appeared sporadically. In the sketchbooks of Goya (1746–1828), that compassionate connoisseur of deformity, there is a lovely sepia-wash portrait of a young mother proudly displaying her deformed child to two inquisitive old women. And there are, scattered throughout the early teratological literature, any number of people with the disorder. In his Tabulae (1844–49), Willem Vrolik gave a portrait of a phocomelic, a famous eighteenth-century Parisian juggler, Marc Cazotte, also known as ‘Le Petit Pepin’. Vrolik also shows Cazotte’s skeleton, which still hangs in the Musée Duputryen in Paris, though its legs, by sad irony, are now missing. These cases of phocomelia might have been caused by some chemical or other, but they may also have been due to mutations, several of which cause the disorder. But until the 1960s, phocomelics were rare, little more than anatomical curiosities. Thalidomide turned them into icons of medical hubris.
PHOCOMELIA. MARC CAZOTTE, A.K.A. PEPIN (1757–1801). FROM WILLEM VROLIK 1844–49 TABULAE AD ILLUSTRANDAM EMBRYOGENESIN HOMINIS ET MAMMALIUM TAM NATURALEM QUAM ABNORMEM.
How does thalidomide have its devastating effects? A comprehensive bibliography on the chemical and its consequences would run to about five thousand technical papers, but for all that, thalidomide is still poorly understood. Some things are clear. It is a teratogen and not a mutagen: the children of thalidomide victims are at no greater risk of congenital disorders than any others. Instead thalidomide inhibits cell proliferation. Taken by a pregnant woman during the time when she is most susceptible to morning sickness (thirty-nine to forty-two days after conception), it circulates throughout the bodies of mother and child and stops cells from dividing. This is when the earliest populations of cells that will form each part of the infant’s future limbs are establishing themselves. Depending on the exact duration of the exposure, the precursors of one or more bones will fail to multiply; the result is a limb with missing parts. It is even thought that thalidomide may impede, quite directly, the fibroblast growth factors that are so essential to limb-bud development, but this remains speculation. Whatever its exact modus operandi, thalidomide is clearly a powerful drug and so a perennially attractive one. The taboo that surrounds it is breaking down as proposals for its use against a variety of diseases proliferate. In South America it is used to treat leprosy. Inevitably, infants with limb deformities are appearing once again as it is given to women who do not know that they have conceived.
GOING DIGITAL
Metric, with its base 10 units, exists only because the savants of the Académie Française who devised the system had ten fingers each on which they presumably learned to count. If pigs could do mathematics, they would probably measure their swill using a Système International devised from base 8, for they have only four digits per hoof. Horses have one digit per limb, camels have two, elephants have five, but guinea pigs have four on the fore-limbs and three behind. Cats and dogs have five on the forefeet and five on the hind feet, but one of those is small, and is called a ‘dew-claw’. Apart from some frogs and a kind of dolphin called a vaquita, most vertebrates never have more than five digits per limb.
Why this is so is deeply obscure. It is not as though extra digits are impossible to make. Mammals of all sorts sometimes show extra digits, but they are never common. St Bernards, Great Pyrenees, Newfoundlands and other large dogs are especially prone to having six digits on each foot – the duplication being an extra dew-claw. Ernest Hemingway’s cats were polydactylous, and their many-toed descendants still live in the grounds of his Key West house. Fifteen per cent of the feral cats of Boston are polydactylous (some have up to ten extra toes), but there are no feral polydactylous cats in New York. There are many polydactylous strains of mice: one is called Sasquatch in homage to Big Foot, but most have more prosaic names such as Doublefoot or Extra-Toes. The American geneticist Sewall Wright once produced a baby guinea pig with forty-four fingers and toes in all, but it did not live.
And many people are born with extra digits. About i in 3000 Europeans is born with extra fingers or toes (or both), and about 1 in 300 Africans. Any digit can be duplicated, but in Africans it usually a little finger (pinkie), while in Europeans it tends to be a thumb. Polydactyly is usually genetic, frequently dominant, and can run for many generations in families. Long before Gregor Mendel ever lived, the French mathematician Pierre-Louis Moreau de Maupertuis (1698–1758) described the inheritance of Polydactyly in the ancestors and descendants of a Berlin physician called Jacob Ruhe. Ruhe’s grandmother had six fingers on each hand and six toes on each foot, as did his mother, as did he and three of his seven siblings, and two of his five children. Others have claimed even more impressive polydactylous pedigrees. In 1931 the Russian geneticist E.O. Manoiloff published an account of a polydactylous Georgian, Via?eslav Michailovi? de Camio Scipion, who, he said, was able to document his descent from a lineage of polydactylous forebears reaching back six centuries.
If the apical ectodermal ridge ensures that our limbs grow out into space, another equally unobtrusive piece of limb-bud ensures that we have the right number and kinds of fingers. It was again John Saunders, along with a collaborator, Mary Gasseling, who discovered it. They found that if they transplanted a piece of mesoderm from the tailmost edge of one chicken limb-bud onto the headmost edge of another (so that the bud had two tailmost edges in opposite orientation to each other), the result was a chicken wing with twice the usual number of digits. Most remarkably of all, the experimental wings were like a particularly exotic variety of polydactyly in humans. They resembled people who, far from having just an extra digit or two, have hands and feet that are almost completely duplicated with up to ten digits each. The polydactylous wings had a peculiar mirror-image geometry, one shared by duplicated hands in humans. If each finger is given a code in which the thumb is 1, forefinger 2, index-finger 3, ring-finger 4, and pinkie 5, then a normal, five-fingered, hand has the formula ‘12345’, while a duplicated hand has the formula ‘5432112345’. It is that strangest of things, an anatomical palindrome.
MIRROR-IMAGE POLYDACTYLY. LEFT HAND OF A WOMAN WITH EIGHT DIGITS. FROM WLLLIAM BATESON 1894 MATERIALS FOR THE STUDY OF VARIATION.
Saunders and Gasseling called their potent piece of mesoderm the ‘zone of polarising activity’ or ‘ZPA’. It is thought to be the source of a morphogen. At its source, where it is most concentrated, this morphogen induces naive mesoderm to become the little finger; further away, lower concentrations induce the ring, index, and forefinger in succession, and at the far opposite end of the limb, you get a thumb.
This account of how most of us come by our five fingers brings to mind the organiser. Like the organiser, the ZPA has the uncanny ability to impose order on its surroundings. And, just as the organiser morphogen was so eagerly sought for so long, so too, in recent years, has been the morphogen of the ZPA. It is almost certainly a signalling protein, likely a familiar one, a member of one of the great families of signalling proteins that also work elsewhere in the embryo. But limb-buds contain a plethora of such proteins, and it is hard to know which of them is the morphogen itself. In the past few years, several candidate molecules have been said to fit the bill. One of them is sonic hedgehog.
Sonic hedgehog appears in the limb-bud precisely where one would expect a morphogen to be: only in the mesoderm of the tailmost edge, exactly coincident with Saunders and Gasseling’s ZPA. It also does what one would expect a morphogen to do: shape limbs. Chicken wings can be sculpted into new and improbable forms – including duplicate mirror-image polydactylous ones – simply by manipulating the presence of sonic in the bud. And then there are the mutants. Mutations in at least ten genes cause Polydactyly in humans and all seem to affect, in some way or other, sonic’s role in the limb.
But, as we saw in the previous chapter, sonic hedgehog does not just determine how many fingers and toes we have. It also divides our brains, decides how widely spaced our eyes will be, and regulates much else beside
s. It is an incorrigibly promiscuous molecule. Could we see the pattern of the sonic hedgehog gene’s activity over time, as in time-lapse photography, we would see it flashing on and off throughout the developing embryo and foetus, now in this incipient organ, now in that one.
The devices responsible for all this have a formidable task, and nowhere, given sonic’s power to direct the destiny of cells, do they have much room for error. These devices are transcription factors or ‘molecular switches’. Some of them keep sonic in check. Should they be disabled by mutation, sonic turns on in parts of the limb-bud that it otherwise would not – and the result is extra fingers and toes. Other mutations do not disable the transcription factors themselves, but rather delete the regulatory elements to which they bind. The result, however, is the same: a confusion of morphogen gradients and an embarrassment of digits.
Polydactyly mutations relax control of sonic hedgehog altering the balance of power in favour of ubiquity. But other mutations have exactly the opposite effect and prevent sonic from appearing in the limb-bud at all. The most blatant example of such a mutation is, of course, one that disables the sonic gene itself. Sonic-less mice have, in addition to their many other defects, no paws. This is strikingly reminiscent of a disorder that we have already come across: acheiropody, the disorder of the aleijadinhos. Indeed, there is some (disputed) evidence that the acheiropody mutation disables a regulatory element essential to sonic’s presence in the limb.
This catalogue of mutations only hints at the complexities of gene regulation in the embryo. Whether or not a gene is turned on in a given cell depends on what transcription factors are found in that cell’s nucleus, and their presence depends on the presence of yet other transcription factors, and so on. At first glance hierarchies of this sort seem to involve us in an infinite regression in which the burden of producing order is merely placed upon a previous set of entities which must, themselves, be ordered. But this dilemma is more apparent than real. The embryo’s order is created iteratively. Sonic’s precise presence in the ZPA is defined in part by the activity of Hox genes in the trunk mesoderm from which limbs grow. But the geometrical order that these genes give to the limb is crude; sonic’s task is to refine it further. Beyond sonic there are, of course, yet further levels of refinement in which order is created on ever smaller scales, and each of them requires subtle and interminable negotiations, the nature of which we scarcely understand.