Mutants (6 page)

But where are the origins of conjoined twins to be found if not in partially split embryos? Sir Thomas Browne called the womb ‘the obscure world’, and so it is – never more so than when we try to explain the creation of conjoined twins. The latest ideas suggest, however, that Aristotle’s dichotomy – fission
or
fusion – is illusory. The making of conjoined twins is, first, a matter of making two embryos out of one, and then of gluing them together. Moreover, the way in which two embryos are made out of one is nothing so crude as some sort of mechanical splitting of the embryo. It is, instead, something more subtle and interesting. Indeed, although we perceive conjoined twins as the strangest of all forms that the human body can take (as recently as 1996
The Times
referred to one pair of twins as ‘metaphysical insults’), they have shown us the devices by which our bodies are given order in the womb.

ORGANISE ME

On the seventh day after conception, a human embryo begins to dig. Though only a hollow ball made up of a hundred or so cells, it is able to embed itself in the uterine linings of its mother’s womb that are softened and swollen by the hormones of the
menstrual cycle. Most of the cells in the hollow ball are occupied with the business of burrowing, but some are up to other things. They are beginning to organise themselves into a ball of their own, so that by day 9 the embryo is rather like one of those ingenious Chinese toys composed of carved ivory spheres within spheres within spheres. By day 13 it has disappeared within the uterine lining, and the wound it has caused has usually healed. The embryo is beginning to build itself.

Its first task is to make the raw materials of its organs. We are three-dimensional creatures: bags of skin that surround layers of bone and muscle that, in turn, support a maze of internal plumbing; and each of these layers is constructed from specialised tissues. But the embryo faces a problem. Of the elaborate structure that it has already built, only a minute fraction – a small clump of cells in the innermost sphere – is actually destined to produce the foetus; all the rest will just become its ancillary equipment: placenta, umbilical cord and the like. And to make foetus out of this clump of cells, the embryo has to reorganise itself.

The process by which it does this is called ‘gastrulation’. At about day 13 after conception, the clump of cells has become a disc with a cavity above it (the future amniotic cavity) and a cavity below it (the future yolk sac). Halfway down the length of this disc, a groove appears, the so-called ‘primitive streak’. Cells migrate towards the streak and pour themselves into it. The first cells that go through layer themselves around the yolk cavity. More cells enter the streak and form another layer above the first. The result is an embryo organised into three layers where once there was one: a gastrula.

The three layers of the gastrula anticipate our organs. The top layer is the ectoderm – it will become the outer layers of the skin and most of the nervous system; beneath it is the mesoderm – future muscle and bone; and surrounding the yolk is the endoderm – ultimate source of the gut, pancreas, spleen and liver. (
Ecto-, meso-
and
endo-
come from the Greek for outer, middle and inner
derm
– skin – respectively.)

The division sounds clear-cut, but in fact many parts of our bodies – teeth, breasts, arms, legs, genitalia – are intricate combinations of ectoderm and mesoderm. More important than the material from which it builds its organs, the embryo has also now acquired the geometry that it will have for the rest of its life. Two weeks after
egg
met sperm, the embryo has a head and a tail, a front and a back, and a left and a right. The question is, how did it get them?

In the spring of 1920, Hilda Pröscholdt arrived in the German university town of Freiburg. She had come to work with Hans Spemann, one of the most important figures in the new, largely German, science of
Entwicklungsmechanik
, ‘developmental mechanics’. The glassy embryos of sea urchins were being bisected; green-tentacled
Hydra
lost their heads only to regrow them again; frogs and newts were made to yield up their eggs for intricate transplantation experiments. Spemann was a master of this science, and Pröscholdt was there to do a Ph.D. in his laboratory. At first she floundered; the experiments that Spemann asked her to do seemed technically impossible and, in retrospect, they were. But she was bright, tenacious and competent, and in
the spring of 1921 Spemann suggested another line of work. Its results would provide the first glimpse into how the embryo gets its order.

Then as now, the implicit goal of most developmental biologists was to understand how human embryos construct themselves, or failing that, how the embryos of other species of mammal do. But mammal embryos are difficult to work with. They’re hard to find and difficult to keep alive outside the womb. Not so newt embryos. Newts lay an abundance of tiny eggs that can, with practice, be surgically manipulated. It was even possible to transplant pieces of tissue between newt embryos and have them graft and grow.

The experiment which Spemann now suggested to Hilda Pröscholdt entailed excising a piece of tissue from the far edge of one embryo’s blastopore – the newt equivalent of the human primitive streak – and transplanting it onto another embryo. Observing that the embryo’s tissue layers and geometry arose from cells that had passed through the blastopore, Spemann reasoned that the tissues at the blastopore’s lip had some special power to instruct the cells that were travelling past it. If so, then embryos that had extra bits of blastopore lip grafted onto them might have – what? Surplus quantities of mesoderm and endoderm? A fatally scrambled geometry? Completely normal development? Earlier experiments that Spemann himself had carried out had yielded intriguing but ambiguous results. Now Hilda Pröscholdt was going to do the thing properly.

Between 1921 and 1923 she carried out 259 transplantation experiments. Most of her embryos did not survive the surgery. But
six embryos that did make it are among the most famous in developmental biology, for each contained the makings of not one newt but two. Each had the beginnings of two heads, two tails, two neural tubes, two sets of muscles, two notochords, and two guts. She had made conjoined-twin newts, oriented belly to belly.

This was remarkable, but the real beauty of the experiment lay in Pröscholdt’s use of two different species of newts as donor and host. The common newt, the donor species, has darkly pigmented cells where the great-crested newt, the host species, does not. The extra organs, it was clear, belonged to the host embryo rather than the donor. This implied that the transplanted piece of blastopore lip had not
become
an extra newt, but rather had
induced
one out of undifferentiated host cells. This tiny piece of tissue seemed to have the power to instruct a whole new creature, complete in nearly all its parts. Spemann, with no sense of hyperbole, called the far lip of the newt’s blastopore ‘the organiser’, the name by which it is still known.

For seventy years, developmental biologists searched in vain for the source of the organiser’s power. They knew roughly what they were looking for: a molecule secreted by one cell that would tell another cell what to do, what to become, and where to go.

Very quickly it became apparent that the potency of the organiser lay in a small part of mesoderm just underneath the lip of the blastopore. The idea was simple: the cells that had migrated through the blastopore into the interior of the embryo were naive, uninformed, but their potential was unlimited. Spemann aphorised this idea when he said ‘We are standing and
walking with parts of our body that could have been used for thinking had they developed in another part of the embryo.’ The mesodermal cells of the blastopore edge were the source of a signal that filtered into the embryo, or to use the term that was soon invented, a
morphogen
. This signal was strong near its source but gradually became fainter and fainter as it dissipated away. There was, in short, a three-dimensional gradient in the concentration of morphogen. Cells perceived this gradient and knew accordingly where and what they were. If the signal was strong, then ectodermal cells formed into the spinal cord that runs the length of our back; if it was faint, then they became the skin that covers our body. The same logic applied to the other germ layers. If the organiser signal was strong, mesoderm would become muscle; fainter, kidneys; fainter yet, connective tissue and blood cells. What the organiser did was pattern the cells beneath it.

It would be tedious to recount the many false starts, the years wasted on the search for the organiser morphogen, the hecatombs of frog and newt embryos ground up in the search for the elusive substance, and then, in the 1960s, the growing belief that the problem was intractable and should simply be abandoned. ‘Science,’ Peter Medawar once said, ‘is the Art of the Soluble.’ But the soluble was precisely what the art of the day could not find.

In the early 1990s recombinant DNA technology was applied to the problem. By 1993 a protein was identified that, when injected into the embryos of African clawed toads, gave conjoined-twin tadpoles. At last it was possible to obtain – without crude surgery – the results that Hilda Pröscholdt had found so
many years before. The protein was especially good at turning naive ectoderm into spinal cord and brain. With a whimsy that is pervasive in this area of biology, it was named ‘noggin’. By this time techniques had been developed that made it possible to see where in an embryo genes were being switched on and off. The noggin gene was turned on at the far end of the blastopore’s lip, just where the gene encoding an organising morphogen should be.

Noggin is a signalling molecule – that is, a molecule by which one cell communicates with another. Animals have an inordinate number of them. Of the thirty thousand genes in the human genome, at least twelve hundred are thought to encode proteins involved in communication between cells. They come in great families of related molecules: the transforming growth factor-betas (TGF-?), the hedgehogs and the fibroblast growth factors (FGFs) to name but a few, and some families contain more than a dozen members. The way they work varies in detail, but the theme is the same. Secreted by one cell, they attach to receptors on the surfaces of other cells and in doing so begin a sequence of molecular events that reaches into the recipient cell. The chain of information finally reaches the nucleus, where batteries of other genes are either activated or repressed, and the cell adopts a fate, an identity.

When noggin was first discovered, it was supposed that its uncanny powers lay in an ability to define the back of the embryo from the front – more precisely, to instruct naive ectodermal cells to become spinal column rather than skin. This was the simplest interpretation of the data. Noggin, the thinking
went, spurred ectodermal cells on to higher things; without it, they would languish as humble skin.

The truth is a bit more subtle. The probability that a cell becomes spinal column rather than skin is not just a function of the quantity of noggin that finds its way to its receptors, but is rather the outcome of molecular conflict over its fate. I said that our genomes encode an inordinate number of signalling molecules. This implies that the cells in our bodies must be continually bathed in many signals emanating from many sources. Some of these signals speak with one voice, but others offer conflicting advice. Noggin from the organiser may urge ectoderm to become neurons, but as it does so, from the opposite side of the embryo another molecule, bone morphogenetic protein 4 (BMP4) instructs those same cells to become skin.

The manner in which the embryo resolves the conflict between these two signals is ingenious. Each signal has its own receptor to which it will attach, but noggin, with cunning versatility, can also attach to free BMP4 molecules as they filter through the intercellular spaces, and disable them. Cells close to the organiser are not only induced to become neurons, but are also inhibited from becoming skin; far from the organiser the opposite obtains. The fate of a given cell depends on the balance of the concentration between the two competing molecules. It is an ingenious device, only one of many like it that work throughout the development of vertebrate bodies, at scales large and small, to a variety of ends; but here the end is a toad or a child that has a front and a back. In a way, the embryo is just a microcosm of the cognitive world that we inhabit, the world of signals that insistently urge us to travel to one
destination rather than another, eschew some goals in favour of others, hold some things to be true and others false; in short, that moulds us into what we are.

It is actually quite hard to prove that a gene, or the protein that it encodes, does what one supposes. One way of doing so is to eliminate the gene and watch what happens. This is rather like removing a car part – some inconspicuous screw – in order to see why it’s there. Sometimes only a rear-view mirror falls off, but sometimes the car dies. So it is with mice and genes. If noggin were indeed the long-sought organising molecule, then any mouse with a defective noggin gene should have a deeply disordered geometry. For want of information, the cells in such an embryo would not know where they were or what to do. One might expect a mouse that grew up in the absence of noggin to have no spinal column or brain, but be belly all round; at the very least one would expect it to die long before it was born. Oddly enough, when a noggin-defective mouse was engineered in 1998, it proved to be really quite healthy. True, its spinal cord and some of its muscles were abnormal, but its deformities were trivial compared to what they might have been.

The reason for this is still not completely understood, but it probably lies in the complexity of the organiser. Since the discovery of noggin at least seven different signalling proteins have been found there, among them the ominously named ‘cerberus’ (after the three-headed dog that guards the entrance to Hades), and the blunter but no less evocative ‘dickkopf’ (German for ‘fat-head’). This multiplicity is puzzling. Some of these proteins
probably have unique tasks (perhaps giving pattern to the head but not the tail, or else ectoderm but not mesoderm), but it could also be that some can substitute for others. Biologists refer to genes that perform the same task as others as ‘redundant’ in much the same sense that employers do: one can be disposed of without the enterprise suffering ill-effects. At least two of the organiser signals, noggin and another called chordin, appear to be partially redundant. Like noggin, chordin instructs cells to become back rather than front, neurons rather than skin, and does so by inhibiting the BMP4 that filters up from the opposite side of the embryo. And, like noggin-defective mice, mice engineered with a defect in the chordin gene have more or less normal geometry, although they are stillborn. However, doubly-mutant mice, in which both the noggin and chordin genes have been disabled, never see the light of day. The doubly-mutant embryos die long before they are born, their geometries profoundly disordered. They can only be found by dissecting the mother in early pregnancy.