Metamorphosis, page 23
What Jim eventually discovered was really quite amazing. Knowing that the only part of its body the mothi could move while metamorphosing was its abdomen, he secured the pupa by waxing one end of a thread to the tip of the abdomen and attaching the other end to a lever that wrote on a slowly revolving drum. That way he could look at the rhythm of abdominal movements over the day-night cycle. For three weeks he carefully recorded five pupae. And while they emerged as adult moths over a period of three days, all five did so in the exact same one-hour window, in the late afternoon. This was thrilling but what came next was truly unbelievable. When Jim removed the brain from these pupae early in development, they survived to complete metamorphosis, emerging as brainless adults—but only at completely random times. If, on the other hand, he implanted a brain into the abdomen of one of these brainless animals, they’d emerge at the appropriate time. He even implanted a brain from a different species into a brainless host only to watch it emerge as a moth precisely at the hour typical of the brain donor species, rather than the host. Incredibly, Jim had moved a biological clock from one animal species to another.
The once-in-a-lifetime event in which a moth emerges from its pupa (called eclosion) had to be controlled by the brain. But while performing his Wigglesworth-like experiments, Jim hadn’t hooked up the nervous systems of the implant and the host, so the timing of the eclosion couldn’t be a neurological event. Rather, it must depend on some secreted molecule, something like a hormone. The search led him to the discovery of a new insect hormone he named eclosion hormone. Incredibly, he could take an extract of this hormone, inject it into the abdomen of a moth that had finished metamorphosis and was waiting for the proper time to emerge, and get the moth to begin its “emergence sequence”: a thirty-minute period of frequent rotary movements of the abdomen followed by a thirty-minute period of quiescence. Then, with repeated contractions of the abdomen in the next thirty minutes, the moth would propel itself forward and out of its old cuticle. Jim saw this same behavior when he manually removed the covering cuticle; the hormone was clearly inducing the behavior necessary for shedding the cuticle, even if there was no cuticle there to shed. Carefully opening up the abdomen of a moth to reveal the underlying chain of four nerve clumps (called ganglia), Jim connected electrodes to a number of motor nerves, added eclosion hormone, and got the same result: The complex one-and-a-half-hour program of behavior was programmed into the central nerve system. Finally, and most incredibly of all, when he cut out the ganglia from the abdomen and just placed them in a dish, the addition of the hormone generated the same ninety-minute program, a precise recapitulation of the behavior shown in the whole animal. The people who had chosen him to be a Harvard junior fellow had made a solid bet.
Meanwhile, shortly after they married, Harvard offered Lynn and Jim a very attractive deal. Sixteen miles from downtown Cambridge, in Bedford, a former military installation used to store Nike missiles had been donated to the university and converted into a field station. A new faculty member named Richard Taylor would be in charge of research; Lynn and Jim could live in the former officer’s quarters, and would be given plenty of space for their own work. Taylor’s vertebrate locomotion studies involved research on a number of exotic animals, and as caretakers and sole human inhabitants of the station, Jim and Lynn would be responsible for feeding and cleaning the cages of cheetahs, chimpanzees, baboons, bush babies, wallaroos, and tenrecs, alongside the more mundane dogs and goats. Taylor lived half a mile from the station and had a small corral and horse stables at the back of his property; if Jim and Lynn wanted to use it, he said, they were welcome. Rekindling Lynn’s childhood love, the couple bought two horses, a happy addition to the menagerie of animals that already surrounded them. The former military installation sported two underground silos. Isolated from the temperature and light cues of the day-night cycle above, they were a perfect place for studying biological clocks.
Back at Harvard, where Jim and Lynn also had labs, an exciting new resident had recently arrived on campus. The tobacco hornworm (Manduca sexta) is a large brown moth that gets its name from the hornlike structure at the rear end of its gigantic caterpillar, and for its ability to sequester and secrete the neurotoxin nicotine from the tobacco plant. Easy to rear in the lab, big enough to dissect, and, most importantly, generating every six weeks as opposed to the silk moth which had only one generation a year, Manduca was a godsend. And since it possessed a diapause determined by recurring cycles of light and dark that could be produced artificially, it was a perfect animal for doing experiments. A student of the Cretan professor Fotis Kafatos named Ray Hakim had been responsible for bringing it to the Harvard Biological Laboratories, but it was Lynn Riddiford who would tame the moth, making it, in her words, the “laboratory rat of insect endocrinology.”
“What questions did you want to answer?” I ask her. Well, how metamorphosis really works. What makes an egg hatch a larva, a larva become a pupa, and a pupa produce an adult? Wigglesworth and Williams had laid out the basic framework of metamorphic control but there remained many holes in it: What were the genes involved? And might hormones work directly upon them? Early work out of a lab in Germanyii seemed to suggest they might. But there were so many questions no one knew how to answer.
Working with Manduca, Lynn soon made an important discovery. When an insect grows, it molts to escape its exoskeleton, a process called ecdysis. The crux of this process is exchanging an old skin for a new one. Since Manduca produced many offspring in a short time, it also produced more mutants; one of these caterpillars was black instead of green. This opened up an amazing opportunity. By placing a small drop of juvenile hormone on the black skin at just the right time, Lynn showed the exposure would create a green spot at the next molt. Next, she exposed a piece of the skin to the molting hormone ecdysone, and implanted it on a larva about to undergo its penultimate molt. (It wasn’t really hard to do, she laughs delightfully, though maybe a bit more tricky than just cutting off heads like Wigglesworth.) After it underwent its final molt, Lynn recovered the implant (easy to do, again, since thanks to the black mutant it was of a different color). What she wanted to know was whether the epidermis had turned into a larval or pupal cuticle. If it had turned into a pupal cuticle, that would mean that the exposure to the hormone had committed it to pupation even though it was on the body of a larva that was still undergoing its penultimate molt. Lo and behold, that’s just what happened. Exposure to the ecdysone hormone had a dramatic effect: In the absence of juvenile hormone, it made a caterpillar larva commit to making a pupa.
Commitment. That was the crux of it. And there was a whole system of cues and clocks and molecules that brought it about. But before Lynn could figure out how the genetic control of metamorphosis worked, Harvard would need to decide whether it was committed to her. As it turned out, it wasn’t. Lynn insists, even today, that it had nothing to do with her gender (Jim: “She doesn’t have a feminist bone in her body!”). When she was appointed assistant professor in 1966, she says, she was told at the outset that she wouldn’t get tenure since there were already too many insect people in the department. She was brought up for tenure, nonetheless, and declined, despite the fact that she already had an impressive four single-authored articles published in Science, and was pulling in good grant money. Whether or not her sex had anything to do with it, she and Jim would need to find a new home.
That’s when they came out west, to Seattle. It wasn’t yet the birthplace of Amazon and Starbucks, and the couple were able to afford a ten-acre farm, where they held three horses and filled their house with cats. Of course, Jim and Lynn brought Manduca with them, and began working at the University of Washington, whose faculty they joined. They decided that they would not have any kids; until and after their retirement, when they moved first to the Janelia Research Campus of the Howard Hughes Medical Institute in Virginia, and later back west to Friday Harbor island, they’d devote their lives to metamorphosis.
When we visit the Friday Harbor Marine Research Station together, I’m struck by the serenity of the place, and its gravitas. The station occupies a cluster of wooden huts on a grassy campus overlooking a San Juan Islands bay, just a short drive from Jim and Lynn’s house. Narrow paths wind through groves of shade trees, past a gleaming sculpture representing the green fluorescent protein (a molecule discovered at the station). An American flag flutters high overhead. The University of Washington, to which the station belongs, kindly allows Jim and Lynn to work in their spacious lab here, for as long as they’d like. “You want to learn what we uncovered about metamorphosis?” Jim asks. “Come, let me tell you the story of the molecular trinity.”
The control of insect metamorphosis had long been a mystery. As with the sea squirt and starfish, there were clearly different genetic programs running at once. If an insect undergoes complete metamorphosis, then its genome contains the instructions for making three distinct body forms: the larva, the pupa, and the adult. But what were the genes that made this system work? From the beginning, and for many decades, clue-by-clue, this would be Lynn’s Holy Grail.
When they were still at Harvard, experimenting in their abandoned missile silos, Jim had shown that there was a “critical period” during the day in which Manduca larvae molted. This had to do with the controlled release of Williams’s “brain hormone” (rechristened the master prothoracicotropic hormone, or PTTH, in 1972), which triggered other hormones down the road. There were three distinct phases in which the insect loosens the connection between its old and new armor, shedding its exoskeleton, expanding, and then hardening a new one. Jim also showed that there was a later “critical period” during the molt in which juvenile hormone prevented the cuticle of the larva from being colored.iii All of this indicated the existence of a clock.
Meanwhile, Carroll Williams and his student Fred Nijhout proved that there was also a “critical weight” above which the larva would undergo metamorphosis on schedule, whether it was fed or starved. Internal worlds—clocks and hormones—were combining with feeding behavior to bring about metamorphosis.
In Seattle, Lynn returned to her skin-grafting experiments, the ones that clarified that after a certain point in time, Manduca became committed to making a pupa. The moment this happened, Jim showed, was during the animal’s transition from feeding to wandering: the moment when it stopped eating tobacco leaves, coated its body with a secretion, and began building a pupa underground. This was correlated with body size, which meant that it had to do with eating. But the clocks and hormones involved suggested that the control was primarily a matter of processes that were internal to the larva: a series of spikes in PTTH release, a reduction in juvenile hormone release, and the accompanying elevation of the molting hormone ecdysone.iv Hormones work by being secreted from glands into the body, reaching target cells, and then latching on to receptors on chromosomes inside those cells’ nuclei that influence the expression of DNA. In this way, hormonal fluctuations could be sensitive both to external environmental cues and to internal clocks, bringing about metamorphosis. But who were the true masters of this affair? Wigglesworth and Williams didn’t know—no one knew—and Lynn would have to gain new skills to find out.
Genetics had come a long way since Mendel. In the first half of the twentieth century, a Kentuckian named Thomas Hunt Morgan had turned his lab at Columbia University into the “Fly Room,” where his team of pioneers fashioned Drosophila—the fruit fly—into the leading model organism of the field. Mendel’s long sought after elements turned out to be actual material things, within the nuclei of cells on chromosomes, and they built and maintained bodies by making proteins. Just around the time when Lynn was falling in love with science as a teenager at her Maine summer camp, James Watson and Francis Crick at Cambridge University, and Rosalind Franklin and Maurice Wilkins at King’s College in London, were figuring out the structure of DNA. That same year, 1962, when Lynn was writing her PhD at Harvard, François Jacob and Jacques Monod in Paris showed how genes are actually turned on and off: Proteins upstream from them on the chromosome bind to receptors that regulate them, and the genes themselves regulate the proteins in turn. That way, when a cell needs to produce a certain protein—for example, in order to make a hormone—it can turn on the right gene, and end production when appropriate. Neither heat nor energy nor a spirit were responsible; in the end the secret of heredity came down to a molecular code. A cell’s machinery could read, replicate, translate, and pass on the code to the next generation. Spemann and Mangold had been right: Building bodies was about control and regulation. Now molecular biologists would need to do the hard work of finding out which genes controlled what traits and how.
Lynn’s generation was the first to step up to the massive challenge. And over time it became apparent that what genes really are and how they work is more complicated than the pioneers of molecular biology first thought. Still, over five decades—including sabbaticals to gain new tools and with the help of colleaguesv—Lynn meticulously put the pieces of the metamorphosis puzzle together. It was especially crucial that the lab switched from working on Manduca to the Drosophila fly, which had been well characterized as a biological system since the early days in Morgan’s “Fly Room.” There were already nearly two million lines of Drosophila available, with tools to knock almost any of its genes out, or alter their expression, as Lynn wished.
It was toward the end of the 1990s that Lynn’s journey took its first dramatic turn. That’s when she finally found a protein that turns on the gene responsible for an insect’s transformation from a larva to a pupa. Since the protein controls the rate of transcribing information from DNA to messenger RNA (a molecule that helps translate the information in the DNA into a protein) it is called a transcription factor, and this particular transcription factor was called broad. Lynn was never able to find the elusive juvenile hormone receptor that got this whole cascade rolling (two former post-docs eventually did),vi but a researcher named David Martín working in collaboration with Xavier Bellés in Barcelona showed that a second transcription factor, ecdysone-inducible protein or E93 for short, controlled the gene responsible for turning a pupa into an adult, in cockroaches, beetles, and flies. Step-by-hard-earned-step, the gene hunters were inching closer to the holy grail.
As it turned out, a forgotten paper authored by Carroll Williams and Fotis Kafatos in 1971 had postulated three master genes that could be responsible for the three stages—larva/pupa/adult. In the very same genome, the two men had speculated, three separate developmental controls could have evolved to influence radically different traits in one part of the life cycle without influencing traits in another. A researcher named Nancy Moran later wrote about how such “adaptive decoupling” could explain complex life cycles. And while Lynn and Jim remained oblivious to the Moran paper from 1994, and the field had completely forgotten the Williams-Kafatos paper from 1971, the search continued. It was two down, one to go.
Soon a red herring presented itself. The small community of metamorphosis experts became convinced that the third master gene was a gene called Krüppel homolog 1, but Jim and Lynn were skeptical. When a molecular cousin of broad, which was known to play a role in determining the different types of adult fly brain cells, presented itself, the penny dropped. The cousin was chinmo (so called since it induced chronologically inappropriate morphogenesis in an embryo), and like many other genes, it had more than just one role. What Jim and Lynn figured out was that if the nervous system transitions from chinmo to broad to get brain development right, maybe it makes the same switch to move from larva to pupa. It was a good idea but seemed impossible to prove: Repressing the expression of chinmo leads to death of the embryo, so how would one uncover its role in metamorphosis? To get around this problem, Lynn and Jim found a brilliant solution: By removing chinmo from just the front, and not the back, halves of larval segments and imaginal discs, they could compare cells that lacked it with their normal neighbors.
Jim and Lynn with moths in the late 1990s. Courtesy of Jim Truman.
Just a year after the appearance of Martín’s discovery of E93, at seventy-seven and eighty-six years of age respectively, Jim and Lynn published an elegant paper in the Proceedings of the National Academy of Sciences. Considering the chain going back through Wigglesworth to Swammerdam to Harvey and all the way to Aristotle, their accomplishment was nothing short of astonishing: the mystery’s long-awaited crowning moment, the holy grail, brought about by two small-figured people observing the smallest changes in tiny insects with the greatest amount of care. Three master genes—chinmo, broad, E93—orchestrated insect metamorphosis. Jim and Lynn called it “the molecular trinity,” drank a nonalcoholic beer and an orange juice, and took a moment to rest.
Of course, metamorphosis itself was less tidy than the trinity suggested—more like a mosaic than a three-colored flag. Over a long career of experimenting, Lynn and Jim had learned the hard way that the textbook explanation of how juvenile hormone controls metamorphosis—the slogan “Hi-Low-No”—was way too simplistic. In fact, juvenile hormone weaves in and out during the life cycle of a butterfly or moth, and its expression looks like a tapestry. Eyes and wings, legs and abdomens, guts and brains, even different segments of the skin, each mature at different rates. Yes, the general picture is that when juvenile hormone levels are high and chinmo is expressed, broad and E93 are turned off.vii Yes, when PTTH spikes it turns off juvenile hormone and turns on ecdysone, signaling to broad to start the program to pupate. Yes, when pupation is complete, E93 kicks in and shuts down broad, to finally produce an adult. But take the eye, just as one example: While juvenile hormone needs to subside to bring about pupation, if you don’t bring it back for a bit just before the insect sheds its outer cuticle, instead of developing a small pupa eye the pupa will develop an outsized adult eye, like a grotesque mythological monster.viii
