The Gene

by Siddhartha Mukherjee

  To answer these questions, we need to turn the clock of embryological development backward. In 1979, one year after Lewis had published his paper on the genes that govern limb and wing development, two embryologists, Christiane Nüsslein-Volhard and Eric Wieschaus, working in Heidelberg, began to create fruit fly mutants to capture the very first steps that govern the formation of the embryo.

  The mutants generated by Nüsslein-Volhard and Wieschaus were even more dramatic than the ones described by Lewis. In some mutants, whole segments of the embryo disappeared, or the thorax or abdominal compartments were drastically shortened—analogous to a human fetus born with no middle or with no hind segment. The genes altered in these mutants, Nüsslein-Volhard and Wieschaus reasoned, determine the basic architectural plan of the embryo. They are the mapmakers of the embryonic world. They divide the embryo into its basic subsegments. They then activate Lewis’s commander genes to start building organs and body parts in some (and only those) compartments—an antenna on the head, a wing in the fourth segment of the thorax, and so forth. Nüsslein-Volhard and Wieschaus termed these segmentation genes.

  But even the segmentation genes have to have their masters: How does the second segment of the fly thorax “know” to be a thoracic segment, and not an abdominal segment? Or how does a head know not to be a tail? Every segment of an embryo can be defined on an axis that stretches from head to tail. The head functions like an internal GPS system, and the position relative to the head and the tail gives each segment a unique “address” in the embryo. But how does an embryo develop its basic, original asymmetry—i.e., its “headness” versus “tailness”?

  In the late 1980s, Nüsslein-Volhard and her students began to characterize a final flock of fly mutants in which asymmetrical organization of the embryo had been abrogated. These mutants—often headless or tailless—were arrested in development long before segmentation (and certainly long before the growth of structures and organs). In some, the embryonic head was malformed. In others, the front and back of the embryo could not be distinguished, resulting in strange mirror-image embryos (the most notorious of the mutants was called bicoid—literally “two-tailed”). The mutants clearly lacked some factor—a chemical—that determines the front versus the back of the fly. In 1986, in an astonishing experiment, Nüsslein-Volhard’s students learned to prick a normal fly embryo with a minuscule needle, withdraw a droplet of liquid from its head, and transplant it into the headless mutants. Amazingly, the cellular surgery worked: the droplet of liquid from a normal head was sufficient to force an embryo to grow a head in the position of its tail.

  In a volley of pathbreaking papers published between 1986 and 1990, Nüsslein-Volhard and her colleagues definitively identified several of the factors that provide the signal for “headness” and “tailness” in the embryo. We now know that about eight such chemicals—mostly proteins—are made by the fly during the development of the egg and deposited asymmetrically in the egg. These maternal factors are made and placed in the egg by the mother fly. The asymmetric deposition is only possible because the egg itself is placed asymmetrically in the mother fly’s body—thereby enabling her to deposit some of these maternal factors on the head end of the egg, and others on the tail end.

  The proteins create a gradient within the egg. Like sugar diffusing out of a cube in a cup of coffee, they are present at high concentration on one end of the egg, and low concentration on the other. The diffusion of a chemical through a matrix of protein can even create distinct, three-dimensional patterns—like a pool of syrup ribboning into oatmeal. Specific genes are activated at the high-concentration end versus at the low-concentration end, thereby allowing the head-tail axis to be defined, or other patterns to be formed.

  The process is infinitely recursive—the ultimate chicken-and-egg story. Flies with heads and tails make eggs with heads and tails, which make embryos with heads and tails, which grow into flies with heads and tails, and so forth, ad infinitum. Or at a molecular level: Proteins in the early embryo are deposited preferentially at one end by the mother. They activate and silence genes, thereby defining the embryo’s axis from head to tail. These genes, in turn, activate “mapmaker” genes that make segments and split the body into its broad domains. The mapmaker genes activate and silence genes that make organs and structures.I Finally, organ-formation and segment-identity genes activate and silence genetic subroutines that result in the creation of organs, structures, and parts.

  The development of the human embryo is also likely achieved through three similar levels of organization. As with the fly, “maternal effect” genes organize the early embryo into its main axes—head versus tail, front versus back, and left versus right—using chemical gradients. Next, a series of genes analogous to the segmentation genes in the fly initiates the division of the embryo into its major structural parts—brain, spinal cord, skeleton, skin, guts, and so forth. Finally, organ-building genes authorize the construction of organs, parts, and structures—limbs, fingers, eyes, kidneys, liver, and lungs.

  “Is it sin, which makes the worm a chrysalis, and the chrysalis a butterfly, and the butterfly dust?” the German theologian Max Müller asked in 1885. A century later, biology offered an answer. It wasn’t sin; it was a fusillade of genes.

  In Leo Lionni’s classic children’s book Inch by Inch, a tiny worm is saved by a robin because it promises to “measure things” using its inch-long body as a metric. The worm measures the robin’s tail, the toucan’s beak, the flamingo’s neck, and the heron’s legs; the world of birds thus gets its first comparative anatomist.

  Geneticists too had learned the usefulness of small organisms to measure, compare, and understand much larger things. Mendel had shelled bushels of peas. Morgan had measured mutation rates in flies. The seven hundred suspenseful minutes between the birth of a fly embryo and the creation of its first segments—arguably the most intensively scrutinized block of time in the history of biology—had partly solved one of the most important problems in biology: How can genes be orchestrated to create an exquisitely complex organism out of a single cell?

  It took an even smaller organism—a worm of less than an inch—to solve the remaining half of the puzzle: How do cells arising in an embryo “know” what to become? Fly embryologists had produced a broad outline of organismal development as the serial deployment of three phases—axis determination, segment formation, and organ building—each governed by a cascade of genes. But to understand embryological development at the deepest level, geneticists needed to understand how genes could govern the destinies of individual cells.

  In the mid-1960s, in Cambridge, Sydney Brenner began to hunt for an organism that could help solve the puzzle of cell-fate determination. Minuscule as it was, even the fly—“compound eyes, jointed legs, and elaborate behavior patterns”—was much too big for Brenner. To understand how genes instruct the fates of cells, Brenner needed an organism so small and simple that each cell arising from the embryo could be counted and followed in time and space (as a point of comparison, humans have about 37 trillion cells. A cell-fate map of humans would outstrip the computing powers of the most powerful computers).

  Brenner became a connoisseur of tiny organisms, a god of small things. He pored through nineteenth-century zoology textbooks to find an animal that would satisfy his requirements. In the end, he settled on a minuscule soil-dwelling worm called Caenorhabditis elegans—C. elegans for short. Zoologists had noted that the worm was eutelic: once it reached adulthood, every worm had a fixed number of cells. To Brenner, the constancy of that number was like a latchkey to a new cosmos: if every worm had exactly the same number of cells, then genes must be capable of carrying instructions to specify the fate of every cell in a worm’s body. “We propose to identify every cell in the worm and trace lineages,” he wrote to Perutz. “We shall also investigate the constancy of development and study its genetic control by looking for mutants.”

  The counting of cells began in earnest in the early 1970s. First,
Brenner convinced John White, a researcher in his lab, to map the location of every cell in the worm’s nervous system—but Brenner soon broadened the scope to track the lineage of every cell in the worm’s body. John Sulston, a postdoctoral researcher, was conscripted to the cell-counting effort. In 1974, Brenner and Sulston were joined by a young biologist fresh from Harvard named Robert Horvitz.

  It was exhausting, hallucination-inducing work, “like watching a bowl of hundreds of grapes” for hours at a time, Horvitz recalled, and then mapping each grape as it changed its position in time and space. Cell by cell, a comprehensive atlas of cellular fate fell into place. Adult worms come in two different types—hermaphrodites and males. Hermaphrodites had 959 cells. Males had 1,031. By the late 1970s, the lineage of each of those 959 adult cells had been traced back to the original cell. This too was a map—although a map unlike any other in the history of science: a map of fate. The experiments on cell lineage and identity could now begin.

  Three features of the cellular map were striking. The first was its invariance. Each of the 959 cells in every worm arose in a precisely stereotypical manner. “You could look at the map and recapitulate the construction of an organism, cell by cell,” Horvitz said. You could say, “In twelve hours, this cell will divide once, and in forty-eight hours, it will become a neuron, and sixty hours later, it will move to that part of the worm’s nervous system and stay there for the rest of its life. And you’d be perfectly right: The cell would do exactly that. It would move exactly there, in exactly that time.”

  What determined the identity of each cell? By the late seventies, Horvitz and Sulston had created dozens of worm mutants in which normal cell lineages were disrupted. If flies bearing legs on their heads had been strange, then these worm mutants were part of an even stranger menagerie. In some mutants, for instance, the genes that make the worm’s vulva, the organ that forms the exit of the uterus, failed to function. The eggs laid by the vulvaless worm could not leave their mother’s womb, and the worm was thus literally swallowed alive by its own unborn progeny, like some monster from a Teutonic myth. The genes altered in these mutants controlled the identity of an individual vulva cell. Yet other genes controlled the time that a cell divided to form two cells, its movement to a particular position in the animal, or the final shape and size that a cell would assume.

  “There is no history; there is only biography,” Emerson once wrote. For the worm, certainly, history had collapsed into a cellular biography. Every cell knew what to “be” because genes told it what to “become” (and where and when to become). The anatomy of the worm was all genetic clockwork and nothing else: there was no chance, no mystery, no ambiguity—no fate. Cell by cell, an animal was assembled from genetic instructions. Genesis was gene-sis.

  If the exquisite orchestration of the birth, position, shape, size, and identity of every cell by genes was remarkable, then the final series of worm mutants generated an even more remarkable revelation. By the early 1980s, Horvitz and Sulston began to discover that even the death of cells was governed by genes. Every adult hermaphrodite worm has 959 cells—but if you counted the cells generated during worm development, a total of 1,090 cells were actually born. It was a small discrepancy, but it fascinated Horvitz endlessly: 131 extra cells had somehow disappeared. They had been produced during development—but then killed during the maturation of the worm. These cells were the castaways of development, the lost children of genesis. When Sulston and Horvitz used their lineage maps to track the deaths of the 131 lost cells, they found that only specific cells, produced at specific times, were killed. It was a selective purge: like everything else in the worm’s development, nothing was left to chance. The death of these cells—or rather their planned, self-willed suicide—also seemed genetically “programmed.”

  Programmed death? Geneticists were just contending with the programmed life of worms. Was death too controlled by genes? In 1972, John Kerr, an Australian pathologist, had observed a similar pattern of cell death in normal tissues and in cancers. Until Kerr’s observations, biologists had thought of death as a largely accidental process caused by trauma, injury, or infection—a phenomenon called necrosis—literally, “blackening.” Necrosis was typically accompanied by the decomposition of tissues, leading to the formation of pus or gangrene. But in certain tissues, Kerr noted, dying cells seemed to activate specific structural changes in anticipation of death—as if turning on a “death subroutine.” The dying cells did not elicit gangrene, wounds, or inflammation; they acquired a pearly, wilting translucence, like lilies in a vase before they die. If necrosis was blackening, then this was death by whiteout. Instinctively, Kerr surmised that the two forms of dying were fundamentally different. This “controlled cell deletion,” he wrote, “is an active, inherently programmed phenomenon,” controlled by “genes of death.” Seeking a word to describe the process, he called it apoptosis, an evocative Greek word for the falling off of leaves from trees, or petals from a flower.

  But what did these “genes of death” look like? Horvitz and Sulston made yet another series of mutants—except these were not altered in cell lineage, but in patterns of cellular death. In one mutant, the contents of the dying cells could not be adequately fragmented into pieces. In another mutant, dead cells were not removed from the worm’s body, resulting in carcasses of cells littering its edges, like Naples on a trash strike. The genes altered in these mutants, Horvitz surmised, were the executioners, scavengers, cleaners, and cremators of the cellular world—the active participants in the killing.

  The next set of mutants had even more dramatic distortions in patterns of death: the carcasses were not even formed. In one worm, all 131 dying cells remained alive. In another, specific cells were spared from death. Horvitz’s students nicknamed the mutant worms the undead or wombies, for “worm zombies.” The inactivated genes in these worms were the master regulators of the death cascade in cells. Horvitz named them ced genes—for C. elegans death.

  Remarkably, several genes that regulate cell death would soon be implicated in human cancers. Human cells also possess genes that orchestrate their death via apoptosis. Many of these genes are ancient—and their structures and functions are similar to those of the death genes found in worms and flies. In 1985, the cancer biologist Stanley Korsmeyer discovered that a gene named BCL2 is recurrently mutated in lymphomas.II BCL2, it turned out, was the human counterpart to one of Horvitz’s death-regulating worm genes, called ced9. In worms, ced9 prevents cell death by sequestering the cell-death-related executioner proteins (hence the “undead” cells in the worm mutants). In human cells, the activation of BCL2 results in a cell in which the death cascade is blocked, creating a cell that is pathologically unable to die: cancer.

  But was the fate of every cell in the worm dictated by genes, and only genes? Horvitz and Sulston discovered occasional cells in the worm—rare pairs—that could choose one fate or another randomly, as if by coin flip. The fate of these cells was not determined by their genetic destiny, but by their proximity to other cells. Two worm biologists working in Colorado, David Hirsh and Judith Kimble, called this phenomenon natural ambiguity.

  But even natural ambiguity was sharply constrained, Kimble found. The identity of an ambiguous cell was, in fact, regulated by signals from neighboring cells—but the neighboring cells were themselves genetically preprogrammed. The God of Worms had evidently left tiny loopholes of chance in the worm’s design, but He still wouldn’t throw dice.

  A worm was thus constructed from two kinds of inputs—“intrinsic” inputs from genes, and “extrinsic” inputs from cell-cell interactions. Jokingly, Brenner called it the “British model” versus the “American model.” The British way, Brenner wrote, “is for cells to do their own thing and not to talk to their neighbors very much. Ancestry is what counts, and once a cell is born in a certain place it will stay there and develop according to rigid rules. The American way is quite the opposite. Ancestry does not count. . . . What counts is the interactions with
its neighbors. It frequently exchanges information with its fellow cells and often has to move to accomplish its goals and find its proper place.”

  What if you forcibly introduced chance—fate—into the life of a worm? In 1978, Kimble moved to Cambridge and began to study the effects of sharp perturbations on cell fates. She used a laser to singe and kill single cells in a worm’s body. The ablation of one cell could change the fate of a neighboring cell, she found, but under severe constraints. Cells that had already been genetically predetermined had almost no leeway in altering their destinies. In contrast, cells that were “naturally ambiguous” were more pliant—but even so, their capacity to alter their destiny was limited. Extrinsic cues could alter intrinsic determinants, but to a point. You could whisk the man in a gray flannel suit off the Piccadilly line and stuff him on the Brooklyn-bound F train. He would be transformed—but still emerge from the tunnels wanting beef pasties for lunch. Chance played a role in the microscopic world of worms, but it was severely constrained by genes. The gene was the lens through which chance was filtered and refracted.

  The discoveries of gene cascades that governed the lives and deaths of flies and worms were revelations for embryologists—but their impact on genetics was just as powerful. In solving Morgan’s puzzle—“How do genes specify a fly?”—embryologists had also solved a much deeper riddle: How can units of heredity generate the bewildering complexity of organisms?

  The answer lies in organization and interaction. A single master-regulatory gene might encode a protein with rather limited function: an on-and-off switch for twelve other target genes, say. But suppose the activity of the switch depends on the concentration of the protein, and the protein can be layered in a gradient across the body of an organism, with a high concentration at one end and a low concentration at the other. This protein might flick on all twelve of its targets in one part of an organism, eight in another segment, and only three in yet another. Each combination of target genes (twelve, eight, and three) might then intersect with yet other protein gradients, and activate and repress yet other genes. Add the dimensions of time and space to this recipe—i.e., when and where a gene might be activated or repressed—and you can begin to construct intricate fantasias of form. By mixing and matching hierarchies, gradients, switches, and circuits of genes and proteins, an organism can create the observed complexity of its anatomy and physiology.