AS the mutant Mariner in the movie Waterworld, Kevin Costner comes complete with a pair of gills. Thanks to a genetic mutation and some suspiciously fast natural selection, the Mariner and his kind have acquired an atavism, a physical trait that in this case vanished from human evolution when our amphibian ancestors turned into reptilian landlubbers some 300 million years ago.
Forget Hollywood. In a developmental biology lab near Washington DC, genetic engineers are creating atavisms for real 鈥 at a fraction of the cost and without the need for special effects. The subjects aren鈥檛 human, of course, they鈥檙e lab mice. Nor, it has to be said, have Charles Bieberich and his team at the Holland Laboratory in Rocksville, Maryland, created anything as scientifically beguiling as a mammal with gills. But what they have reported is still pretty remarkable. By manipulating genes that control the body plans of embryos, the researchers say that they have created mutant mice with backbones that are 200 to 300 million years out of date.
Bieberich and his colleagues aren鈥檛 the only biologists making such a claim. A week before the mutant mice findings graced the pages of the Proceedings of the National Academy of Sciences in May, researchers at the University of Wisconsin at Madison reported something similar in the journal Nature. This time the mutants were fruit fly embryos that developed with a body plan some 350 to 400 million years out of date. And, were there a prize for resurrecting ancestral characteristics, French molecular biologist Pierre Chambon and his team at the University of Louis Pasteur in Strasbourg would have to be in the running. A couple of years ago, Chambon and his colleagues were manipulating genes in mouse embryos when they created something totally unexpected: mutant mice with ear bones halfway between those of mammals and reptiles. Instead of developing normally, says Chambon, the ears followed a developmental plan dating back to the therapsids: primitive, mammal-like reptiles that lived among the dinosaurs some 200 million years ago.
Advertisement
If this interpretation is right then ingrained in mouse DNA is some kind of 鈥済enetic memory鈥 from the days when mammals and reptiles last shared an ancestor. And that memory can be unlocked, at least in the case of ear structure. But little in biology is simple, and it will be some time before researchers like Bieberich and Chambon understand exactly what their genetic manipulations are telling them about the evolution of animal body plans. Why, and in what sense, for example, do the genes of lab mice 鈥渞emember鈥 how to make reptilian ear bones? Why do some genetic mutations, but not others, unlock such 鈥渕emories鈥? And most important of all: have the researchers truly discovered how to throw evolution into reverse? And if they have can they identify some of the genetic changes that turned fish into amphibians, amphibians into reptiles and reptiles into mammals?
鈥淲e鈥檙e not creating things that are so wild we can鈥檛 interpret them,鈥 says Bieberich. In creating embryos with an ancient body plan, he argues, the researchers are only doing in the lab what sometimes happens naturally -namely, producing 鈥渁tavistic mutations鈥. Hind limbs on whales, snakes with rudimentary limbs, people with 鈥渢ails鈥 and horses with hooves split into three 鈥渢oes鈥 instead of the usual one are among the best documented examples. Another is hypertrichosis, or 鈥渨erewolf disease鈥, which results in excessive hair growth all over the face and upper body.
Such naturally occurring atavisms have intrigued biologists for years. But it鈥檚 only since researchers began creating atavisms in the lab that the astonishing persistence of these 鈥済enetic memories鈥 has become clear. The reason for that persistence, researchers now believe, has a lot to do with the laziness of evolution.
One of the most profound ideas to emerge from developmental biology labs in recent years is that the body plans of all animals, from fruit flies to elephants, are controlled by the same kinds of genes. Instead of inventing a new set of body plan genes for each new type of animal, it seems that natural selection has simply tinkered with an old one, a set known as Hox genes. When the first multicellular animals evolved some 700 million years ago, biologists believe that their body plans were largely the work of a primitive set of Hox genes. They also believe that descendants of these genes have been sculpting the body plans of animals ever since. The evolution of fish into amphibians and amphibians into reptiles and so on must have involved many genetic changes. But researchers like Bieberich believe that elaborations of the ancestral animal body plan 鈥 involving step-by-step mutations and duplications of Hox genes 鈥 were central to these transformations.
Elaborate Hox
And this is where atavisms come in. So far, most of the ancient features that have leapt out of the past into labs have resulted from experiments on Hox genes. 鈥淲e weren鈥檛 looking to create a more ancient body plan,鈥 says Bieberich, just trying to find out what different Hox genes do inside embryos during development. This, however, is no small task since mice and other mammals have 38 different Hox genes. One approach is to make transgenic embryos that lack a certain Hox gene or have an overly active form of it. By monitoring how such embryos develop, whether they have any abnormalities for example, researchers can then discover something about the gene鈥檚 role in shaping the body plan.
For some years biologists have known that development goes awry if you disrupt Hox genes inside embryos. Cells may take on the wrong identity, limbs may grow in the wrong place. Now it鈥檚 clear that something else can happen. Disrupt a Hox gene and you may inadvertently resurrect an ancient body plan. And discovering that, says Bieberich, 鈥渉as been a surprise鈥.
In one experiment, his team changed the behaviour of a gene known as Hoxb-8. In mouse embryos, this gene is normally active in cells in the upper abdomen but dormant elsewhere. Bieberich and his colleagues used genetic engineering to boost the gene鈥檚 activity and extend its range in the embryo. The impact on the body plan was dramatic. In the trunk, flexible, 鈥渘eck-like鈥 vertebrae extended further down the back than usual. And in the normally ribless lower part of the backbone, extra ribs sprouted from vertebrae. Moreover, these extra ribs locked onto the backbone without fusing with it in the usual way: they grew as 鈥渇ree ribs鈥.
Bieberich would argue that all these changes can be explained as revivals of an obsolete body plan. Judging from the fossil record and the skeletons of modern fish, amphibians and reptiles, the ancestors of mammals would have had more ribs; and they would have had a greater number of flexible vertebrae, of the kind we have in our necks. Only as mammals evolved did the backbone become more rigid. 鈥淲hat we did in our transgenic mice,鈥 says Bieberich, 鈥渨as to put back a developmental programme that says 鈥榞row free ribs鈥 and 鈥榖uild less rigid vertebrae鈥.鈥
Critics might see it differently. Perhaps all the researchers did was mess up development. After all, body plans depend on the concerted and finely balanced actions of many genes 鈥 disrupt one of these genes and you may upset that balance. 鈥淭ime and again people suggest that all we鈥檝e done is throw a monkey wrench into the system,鈥 says Bieberich. 鈥淏ut the developmental abnormalities we see are quite specific and consistent with the idea that they are atavisms.鈥
Chambon and his Strasbourg team would agree. Three years ago they created what they describe as mutant mouse embryos with backbones similar to those of primitive, jawless fish. And again, it was disrupting a Hox gene that resurrected the ancient body plan. Before about 430 million years ago, the most advanced ancestors of mammals were fish with inflexible, jawless mouths -direct relatives of today鈥檚 lampreys and hagfish. Then, in a step that enormously expanded the vertebrate lifestyle, fish appeared with hinged jaws for feeding, fighting and 鈥渉andling鈥 objects in the water around them. As these jawed fish evolved, part of the backbone was transformed into a new section of skull. According to the Strasbourg researchers, a simple change in a gene called Hoxd-4 may have invoked this transformation.
In normal mouse embryos, Hoxd-4 is active in cells that form vertebrae in the neck, but dormant in the tissue that makes the back of the skull. To find out why, the researchers altered the status quo with genetic engineering. They activated the Hoxd-4 gene in both regions of mice embryos. As a result, the embryos made backbones that were longer than normal and completely failed to build the back of the skull. In short, the embryos began to resemble jawless fish.
Primitive ears
If hinged jaws were a boon to primitive fish, an acute sense of hearing may have been vital to the survival of primitive mammals. Even as the first mammals were appearing, their therapsid ancestors were becoming extinct, and the dinosaurs which would dominate the planet for the next 150 million years were more ferocious competitors. In dinosaurs, as well as in the ancestors of modern reptiles, sound travelled to the inner ear along a single bone. Mammals, by contrast, have three bones in the middle ear 鈥 a malleus, incus and stapes.
Three bones give better sound amplification than one, but they couldn鈥檛 have evolved overnight. Fossils show that the therapsid ancestors of mammals had ears with an intermediate bone structure. When Chambon and his colleagues disabled a gene known as Hoxa-2 in mouse embryos, part of this intermediate ear plan reappeared. In particular, the mutant mouse embryos grew a small bone in their middle ears that is normally only found in reptiles. The appearance of this so-called quadrate bone was a surprise. It suggested the researchers had resurrected a therapsid-like structure (see 鈥淢aking mutant ears鈥).
Labs specialising in fruit fly genetics have also been communing with ancestral spirits. Normal fruit flies have one pair of wings, but their ancestors had many more. Perhaps these ancestral insects needed extra wings to balance properly. Perhaps they used them as oars for swimming. We may never know. But what is clear is that the embryos of modern fruit flies can be induced to develop those extra wings. In May, Sean Carroll and his colleagues at the Howard Hughes Medical Institute in the University of Wisconsin at Madison described in Nature how they produced mutant fly embryos which began to grow up to nine pairs of wings. And yes, the mutations in question involved Hox genes.
Whether any of these genetic manipulations represent steps back in evolution, however, is an open question. Bieberich is doubtful. 鈥淲e鈥檙e probably not turning on old genes, but taking existing developmental pathways and twisting them into ancient pathways.鈥 Jonathan Slack, a developmental biologist at the University of Bath who studies Hox genes, agrees: 鈥淏y altering these genes you can parallel things that have happened in evolution. But that doesn鈥檛 mean that was how it happened.鈥 Slack also worries about the research creating a false sense of 鈥渆volution happening in jumps鈥. The quadrate bone diminished slowly through therapsids and early mammals, he says. Just because you can make it reappear in a jump, doesn鈥檛 mean it vanished in a jump.
Nevertheless, that you can as much as mimic an obsolete body plan by disrupting one gene in 100 000 is a pretty powerful idea. It makes it hard to argue with the central claim of Hox research 鈥 namely, that stepwise elaborations of a single genetic theme have helped to produce the huge variety of animal body plans visible in the fossil record and in the world today. Take the lab mice with ancestral backbones. The general message here, says Bieberich, has little to do with any one gene or mutation. It鈥檚 that step-by-step narrowings of the areas over which various Hox genes were active inside embryos could have paved the way for the evolution of the mammalian backbone. And evidence from another source points to duplications of Hox genes being important in the evolution of body plans.
Mammals, for example, have four clusters of Hox genes and these came from duplications of a single ancestral cluster. Peter Holland, a zoologist at the University of Reading, believes that these duplications may have been vital to the evolution of vertebrates from more primitive animals. Holland and his team are studying Hox genes in the nearest living relative of vertebrates 鈥 a cigar-shaped, aquatic filter feeder called amphioxus. They have discovered that unlike mammals, amphioxus has only a single cluster of Hox genes. Perhaps going from one Hox cluster to four allowed for the huge increase in complexity in the evolution of vertebrates from spineless ancestors. 鈥淲here you have had gene duplications, you are left with multiple genes performing the same role,鈥 says Holland. This would free the spare copies of Hox genes to adopt new functions.
Embryo look-alikes
Comparing Hox genes from different animals may answer one of the most long-standing mysteries in animal development: why embryos from wildly different species appear to enter a look-alike contest part way through their growth. At a certain point in development few people could spot the difference between a fish and a person. Yet both before and after this stage the embryos grow in very different ways.
We may now know why. In what鈥檚 tantamount to a genetic ID for the animal kingdom, clusters of Hox genes show similar patterns of activity in all the embryos studied so far. Starting in and near the head, the genes within a Hox cluster become active one after another in turn as you move closer and closer to the tail 鈥 switching on in sequence like a set of Christmas lights. So while Hox genes may tell cells to do completely different things in different groups of animals, one of their jobs is always to tell cells where they are on the head-to-tail axis of the body.
鈥淥n a very long time scale, I regard the Hox genes as just an abstract ruler,鈥 says Chris Graham, an embryologist at the University of Oxford. Perhaps the need to set up this ruler is why all humans spend a few days looking like fish. Graham, Holland and Slack have pointed out that in all animals studied so far, the stage when the Hox ruler appears is also the stage when embryos look most alike. Maybe, once the Hox ruler evolved to sufficient complexity in each group of animals, the need to set up the Hox code was anchored in that point of embryo development, sheltering it from evolutionary change.
At the same time, it鈥檚 clear that altering Hox genes in lab animals can easily put flesh onto the ghosts of lost body features. And this raises a puzzle. Why isn鈥檛 the history of life on Earth full of ancestral throwbacks, of limbs and bones vanishing only to reappear later in evolution? Natural examples are hard to find, although Carroll and his colleagues point to caterpillars. These creatures may have acquired their unusual abdominal legs, say the researchers, when a Hox mutation revived ancient leg-growing genes that had been switched off earlier in insect evolution. But for most animals, lost features would be of little use. A quadrate bone would not help a mammal to hear. Likewise, snakes are unlikely to walk again. They may have evolved from burrowing lizards but their bodies are now so long that walking (on four legs) would be impossible. As for the Mariner, he should try to keep his head above water. Warm-blooded animals need far more oxygen than a pair of moderately sized gills would provide.
Freak shows?
鈥淪keletal malformations were observed in both forelimbs and hindlimbs 鈥 the ulna and radius were misshapen, the pisiform and triangular carpal bones were fused and abnormal sesamoid bone development occurred.鈥
So runs a typical description in a science journal of the consequences of 鈥渒nocking out鈥 developmental genes in mouse embryos. Most embryos manipulated in this way die before or shortly after birth or are aborted by researchers as soon as all the developmental abnormalities have been recorded: nobody wants any unnecessary suffering. And nobody is out to create mice with messed-up bones and limbs just for kicks. The research is part of an effort to understand how complexes of genes, known in the trade as Hox genes, encode the body plans of mammals. Indeed research into Hox genes has become one of the hottest topics in developmental biology. 杏吧原创s who manipulate animal embryos to find out how the genes work can expect to publish their findings in some of the most prestigious scientific journals.
But however respectable the journal, you can鈥檛 publish papers replete with photos of twisted limb bones and deformed embryos without provoking concern 鈥 not even when the subjects are mice.
Gill Langley, scientific adviser to the Dr Hadwen Trust for Humane Research, calls it 鈥渃uriosity-driven genetic engineering鈥. 鈥溾楬ere鈥檚 a gene, let鈥檚 find out what it鈥檚 doing by knocking it out鈥 is what the researchers are saying 鈥 I find that very worrying.鈥 But researchers say their experiments raise no new moral questions. Geneticists have been manipulating the body plans of fruit flies for years, producing a gallery of weird 鈥 but biologically informative 鈥 mutants. Celebrated examples include mutant flies with eyes on their wings or legs sticking out of their heads.
More recently, advances in genetics have made it possible to disrupt the body plans of mammal embryos. These experiments are 鈥渘o different to any other genetic experiments鈥, says Lewis Wolpert, a developmental biologist at University College London. 鈥淭here鈥檚 nothing special in this respect about Hox genes.鈥 Langley disagrees. 鈥淢ammals are sentient. We don鈥檛 know whether a fruit fly can experience pain or distress, but we do know that mice and other mammals can.鈥
How many mammal embryos are genetically manipulated each year is far from clear. But in Britain, government figures show a sharp increase in the number of transgenic animals being produced in labs and the number of animals bred with harmful defects. 鈥淭he field of genetic engineering is the most rapidly growing area of animal research,鈥 says Donal Crawford, senior researcher at the British Union for the Abolition of Vivisection. 鈥淎nd it鈥檚 the one we are most concerned about.鈥
鈥淎 few years ago things seemed to be changing in a more human and humane direction, but this incredible explosion of genetic engineering has thrown it back 20 years,鈥 says Langley. Researchers are once again seeing animals as mere tools, she claims, and there is a telling resurgence of euphemistic language in scientific papers. Just as civilian deaths become 鈥渃ollateral damage鈥 in warfare, so animals in labs become 鈥渢ransgenic lines鈥, 鈥渓iving materials鈥, or, in one notorious euphemism for transgenic sheep, 鈥渕ammary gland bioreactors鈥.
Debates about the morality of genetically manipulating animals are scarcely new. But to date most arguments have centred on transgenic animals designed for a medical or commercial purpose 鈥 mice with genes that produce human-like diseases, sheep designed to produce expensive drugs, pigs with 鈥渉uman-like鈥 organs for transplantation surgery. Manipulating Hox genes in lab mice might produce academic insights galore. But what other benefits?
鈥淏y understanding how the developmental plan works in animals we gain information that is relevant to humans and human disease,鈥 says Charles Bieberich of the Holland Laboratory in Rockville, Maryland. And Hox genes are no mere curios for genetic engineers, he insists: 鈥淭hey鈥檙e involved in patterning the brain, in cellular responses to hormones and have even been implicated in immune responses.鈥 鈥淲olpert agrees: 鈥淪o fundamental are these genes to biology that it鈥檚 impossible to know where this research will lead.鈥
But if Hox genes are one day destined for medical glory, their star has yet to rise. Of the 2000 or so papers on Hox genes, or their molecular relatives, that have appeared in major scientific journals over the past few years, only one refers explicitly to a human disease.
Not that medical utility is necessary in the eyes of the law. In Britain, researchers who intend to do animal experiments which might lead to 鈥渟uffering, pain, distress or lasting harm鈥 need a licence. But nobody is required to demonstrate a clear medical or commercial purpose. Curiosity is sufficient. (see Diagram)
Making mutant ears
Pierre Chambon and his team at the University of Strasbourg created mice with ancient ear bones by altering the behaviour of a developmental gene.
Fossil evidence suggests that mammals evolved about 200 million years ago from a group of reptiles called the therapsids. The transition involved a virtuoso piece of remodelling that gave mammals a stronger bite and a keener sense of hearing that their reptilian competitors.
The key changes happened at the back of a therapsid鈥檚 jaw, which was used in both chewing and hearing. In hearing, the jaw hinge played the same role as the malleus and the incus in a mammalian ear. Vibration travelled from the eardrum, through the jaw hinge, and then through the stapes to the inner ear. But because the hinge bones had to be big enough to support the action of the jaw, they were too big to efficiently transmit sound. So therapsids did not hear very well.
As mammals evolved, the problem was solved by moving the jaw hinge forwards so that it involved completely different bones. One result was a stronger jaw built from fewer bones. The other was that the old hinge bones were free to shrink to specialise for their role in hearing. The lower bone became the malleus, and the quadrate bone 鈥 the top of the hinge 鈥 became the incus. This didn鈥檛 happen overnight. The very first mammals had two jaw hinges. They used both the old reptilian hinge and the new mammalian hinge side-by-side.
What happens in Chambon鈥檚 mutant mice? Whether haddock or human, early in its life a vertebrate embryo has a primitive head, a tail and about six bulges on the head and neck that look like gills. In fish, most of these bulges, or 鈥減haryngeal arches鈥, end up as gills. But in mammals, evolution has directed the arches along other developmental pathways. Some of the cells from the top two arches develop into parts of the mammalian ear.
The research focused on Hox genes, widely known to be central to the way cells in different parts of an embryo develop. In mammals, the second pharyngeal arch has active Hox genes, but not so the first arch. To discover why, Chambon鈥檚 group switched off one of the genes, Hoxa-2. As a result, the Hox code became scrambled and the second arch behaved more like a first arch. But what the researchers couldn鈥檛 have guessed was that the second arch would follow a first arch plan obsolete for about 200 million years.
The most startling effects of switching off Hoxa-2 are on the three bones that carry sound from the eardrum, through the middle ear, to the fluid-filled inner ear. In normal mice, the first arch forms the first two of these bones, the malleus and the incus, and the second arch builds the third bone, the stapes. In the mutant mice, however, the stapes fails to form and instead the second arch makes an extra malleus and incus, as though it is following a first arch programme. Astonishingly, the second arch also produces the quadrate bone that is normally found at the top of a reptile鈥檚 jaw hinge. (see Diagram).
