# News this Week

Science  07 Jun 2013:
Vol. 340, Issue 6137, pp. 1148
1. # Around the World

1 - Florence, Italy
Three MERS Cases in Italy
2 - Tokyo
Joining the Fight Against Neglected Diseases
3 - Paris
Can Science Help France's Economy?
4 - Oregon and Berlin
Furor Over U.S. GM Wheat

## Florence, Italy

### Three MERS Cases in Italy

A deadly new virus that originated in the Middle East has made another jump to Europe. Italian health authorities have reported three cases of Middle East respiratory syndrome (MERS), the first of whom was a 45-year-old man who fell ill after a 40-day trip to Jordan, where he is assumed to have picked up the virus. A 2-year-old girl and a 42-year-old woman who were in close contact with the man became infected in Italy.

The cluster is another clear sign that the virus can spread between people, although not very efficiently so far. That's worrisome to scientists and public health experts, who fear that MERS may start spreading more rapidly and trigger a pandemic. Caused by a distant cousin of the SARS virus, MERS is known to have occurred in Jordan, Saudi Arabia, Qatar, and the United Arab Emirates; people infected in those countries had previously brought the virus to the United Kingdom, Germany, France, and Tunisia. So far, there have been 53 recorded cases, including 30 deaths.

## Tokyo

### Joining the Fight Against Neglected Diseases

Japan is joining global efforts to contain malaria, tuberculosis, and a variety of tropical diseases in a big way. On 1 June, a recently formed public-private partnership announced agreements to screen tens of thousands of drug candidates from Japanese private and public sector compound libraries for treatments for illnesses that primarily afflict the poor in developing countries.

The 11 initial agreements are the first fruits of a recently formed public-private Global Health Innovative Technology Fund (GHIT Fund), which brings together Japan's foreign affairs and health and welfare ministries, five pharmaceutical companies, and the Bill & Melinda Gates Foundation. The Japanese government is putting up a bit over one-half of the $100 million committed to GHIT over the next 5 years; the drugmakers and the Gates Foundation are contributing the rest. Even though Japan is a major producer of new pharmaceuticals, the country has been a bit behind other nations in contributing to the global health R&D effort, says BT Slingsby, the fund's CEO and executive director. GHIT is working with established nonprofits—the Global Alliance for TB Drug Development, the Medicines for Malaria Venture, and the Drugs for Neglected Diseases initiative—to help develop candidate drugs. http://scim.ag/GHITJapan ## Paris ### Can Science Help France's Economy? On 28 May, France's National Assembly approved a law that aims to simplify the national landscape for research and higher education, making it more efficient and more competitive at the European level. The bill, which comes hand in hand with a new strategic plan called France Europe 2020, also gives the government a greater role in coordinating research. The bill and the strategic plan have been sharply criticized by various groups of researchers and university professors. The new national priorities include health, food security, climate change, sustainable energy, urban systems, digital technologies, and space; they will be periodically revised by a newly created strategic research council. The strategic plan is partly designed to reinvigorate industry through pathbreaking areas such as nanotechnologies and by promoting industry-academia partnerships. But trade unions say that the government can't make science responsible for rescuing the economy and worry that the new plan will erode basic research. "There is a big concern because [the government] wants research to solve an economic problem and an industry problem," says Patrick Monfort, a marine ecologist with CNRS in Montpellier and the general secretary of SNCS-FSU, the national trade union for scientific researchers. http://scim.ag/Franceecon ## Oregon and Berlin ### Furor Over U.S. GM Wheat News that genetically modified wheat plants—last deliberately planted years ago—were found growing on an Oregon farm touched off an international uproar last week. Japan postponed wheat imports from Oregon, while South Korea and the European Union called for stepped up testing to ensure that GM wheat hasn't entered the food supply. Oregon's wheat crop, valued at up to$500 million a year, now stands in jeopardy.

The latest GM row touched off in April after a farmer noticed wheat plants growing on his farm, although it had been sprayed with enough of the herbicide glyphosate to kill normal wheat. He contacted researchers at Oregon State University, Corvallis, who tested plant samples and discovered that they contain an introduced gene for glyphosate resistance, a GM technology field-tested by Monsanto in Oregon and 15 other states from 1998 to 2005. Monsanto dropped the project when it became clear there was little market for GM wheat.

Monsanto has come to a similar conclusion in Europe: It will not apply for new E.U. product licenses for GM varieties or conduct new field trials, a spokesperson told German newspaper Die Tageszeitung last week.

2. # Random Sample

## ScienceLive

Join us on Thursday, 13 June, at 3 p.m. EDT for a live chat with experts on bioelectronics. http://scim.ag/science-live

## Frown for the Camera

For decades, psychologists have used "Pictures of Facial Affect" (conceived by psychologist Paul Ekman) to examine human responses to "universal" facial expressions and emotions: anger, fear, sadness, happiness, surprise, and disgust. But these photos have a drawback: They're primarily of Caucasian adults, and reactions to the expressions can depend on both race and age, says psychologist Vanessa LoBue of Rutgers University, Newark, in New Jersey.

So over the last 5 to 6 years, LoBue, with the help of photographer and former research assistant Cat Thrasher, has created her own set of more than 1200 photos, featuring 190 children of multiple demographics and ages (including the 4- to 6-year-olds shown here). Last month, LoBue received a National Science Foundation grant to conduct validation experiments of her new photo set, in which adults and children of different age groups will try to identify the expressions in each photo, and each photo will then get a "validity score" that researchers can use to design their own experiments. "Ultimately, the goal is to release the set for anyone to use for free," LoBue says. "We spent so many years working on it, we feel like anyone should be able to use it."

## Red Meets White Atop Mont Blanc

By the time this issue of Science appears, 25 Dutch scientists and volunteers should be climbing Europe's highest mountain, Mont Blanc in the French Alps, seeking to better understand the effects of hypoxia and low blood oxygen levels on blood coagulation. The mission, called "the red meets white study," was scheduled to begin on 5 June in the French village of Chamonix near the base of the mountain (expedition logo, inset). Led by Dutch mountain guide Edward Bekker, the climbers plan to attain the summit, 5000 meters above sea level, by 14 June. Every thousand meters, each climber will offer up a drop of blood.

Previous alpine expeditions have sought a direct link—if one exists—between lowered blood oxygen levels and an increased risk of thrombosis, the formation of blood clots within blood vessels. But those efforts were unsuccessful, due to the failure of regular blood coagulation tests at high altitudes: At lower atmospheric pressures, not enough blood enters the catalyst-filled test tubes to get conclusive results.

But, expedition leader biochemist Bas de Laat of Maastricht University has a secret weapon—a new coagulation test specially adapted for the expedition that requires just a single drop of blood. De Laat divided his team into two groups—one hiking and one taking a cable car—to track how physical activity affects coagulation. Then, each team's medical staff, consisting of cardiologists, a hematologist, and anesthesiologists, will use the new test, adapted from a standard test by Synapse BV (a Maastricht University spinoff company of which de Laat is the current CEO) that measures thrombin, a key blood clotting component that forms within 10 minutes after coagulation begins.

3. # Newsmakers

## Storm Chasers Killed in Tornado

Tim Samaras, 55, an engineer and tornado researcher and "storm chaser," was one of 13 people killed by a cluster of tornadoes that swept through central Oklahoma on 31 May. Two other storm chasers—Samaras's son Paul, 24, and researcher Carl Young, 45, were also killed, in the first known fatalities of storm interceptors during a tornado.

Samaras designed and built weather probes that he deployed in the path of tornadoes in order to gain scientific insight into their inner workings. He founded the Tactical Weather Instrumented Sampling in/near Tornadoes Experiment, consisting of a caravan of vehicles equipped with thermodynamic and video probes that deployed each spring during tornado season.

"We are terribly saddened by this news," said a spokesperson for the National Oceanic and Atmospheric Administration in a statement on 3 June. "Samaras was a respected tornado researcher and friend of NOAA who brought to the field a unique portfolio of expertise in engineering, science, writing and videography."

## First Fresenius Award to Immunologist

Yale University immunologist Ruslan Medzhitov has been awarded the first Else Kröner Fresenius Immunology Award. The scientist will receive $650,000 for past achievements, plus an additional$4.5 million for his ongoing research. The Fresenius Foundation plans to award the prize, which commemorates the 25th anniversary of the death of German pharmaceutical entrepreneur Else Kröner, every 4 years; each award will single out promising research in a different field.

"We wanted to single out the most innovative work in immunology done in the past that promises to have the highest impact on future clinical immunology," said immunologist Stefan Kaufmann, head of the selection committee, in a statement.

Medzhitov, who was born in Tashkent, Uzbekistan, worked with Yale immunologist Charles Janeway to elucidate how the human body fights infectious agents. Together, they showed that molecules called toll-like receptors recognize pathogens in the human body and activate the immune system. Medzhitov was controversially left out when the Nobel Committee recognized work in this field in 2011.

## New Award to Target Young Scientists

Ukrainian billionaire Leonard Blavatnik and the New York Academy of Sciences (NYAS) this week announced the creation of a new national award specifically geared to help young scientists still working to establish their careers. Three unrestricted cash prizes of \$250,000 in three categories (physical sciences and engineering, chemistry, and life sciences) will be awarded annually by NYAS and the Blavatnik Family Foundation.

The award grew from a regional awards program that, beginning in 2007, recognized young scientists in New York, New Jersey, and Connecticut. The scientific advisory council for the national award includes a number of heavy-hitters, including Ellis Rubinstein, the chair and CEO of the NYAS, and Tim Appenzeller, incoming news editor of Science. Institutions can choose nominees for the 2014 program from October to December 2013.

"The long-term goal of the Awards is to create a pipeline of scientific support, in which established scientists choose the most outstanding young faculty-rank scientists, who then go on to mentor the next generation of would-be scientists and award winners," Rubinstein said in a statement.

4. # Mysteries of Development

1. John Travis

Development is, literally, the journey of a life time, and it is a trip still as mysterious as it is remarkable. Despite new methods to probe how an animal or plant forms from a single cell, biologists have much to learn about the unimaginably complex process. To identify some of the field's persistent riddles, Senior Editors Beverly Purnell and Stella Hurtley and the news staff of Science have consulted with developmental biologists on our Board of Reviewing Editors and elsewhere. The mysteries offered here are a humbling reminder that our knowledge of development remains to a great extent embryonic.

5. Mysteries of Development

# How Do Organs Know When They Have Reached the Right Size?

1. Gretchen Vogel

Developmental biologists have found dozens of proteins and genes that play a role in the growth of plants and animals, such as imaginal discs and Hippo and morphogenetic proteins, but not what determines organ size.

In the 1920s, biologists began exploring a new way of studying development: surgically removing rudimentary tissues that would form organs and limbs from embryos of one species and transplanting them into those of a related species. In one visually striking example, Yale University zoologists Victor C. Twitty and Joseph L. Schwind removed embryonic tissue that would become a leg in the large salamander species, Ambystoma tigrinum, and transplanted it into the embryo of a smaller species, Ambystoma punctatum. Despite the early stage of the transplant—before limb buds even appear in the subsequent larvae—the legs grew to the size that they would have on their original body; small salamanders ended up with a longer-than-normal leg, and large salamanders with a short leg. The result, published in 1931 and considered a classic experiment today, suggested that something intrinsic in the leg, rather than signals from the rest of the body, determines a limb's final size.

Since then, developmental biologists have found dozens of proteins and genes that play a role in the growth of plants and animals. But how growing organs and organisms can sense their size and know when to stop is still a mystery.

Developmental biologists continue to explore that mystery today, although most of their experiments now use fruit flies instead of salamanders. The current objects of their attention are imaginal discs, flattened sacs of cells that grow during the fly's larval stages. During the pupal stages and morphogenesis, specific discs differentiate to form the adult wings, legs, eyes, antennae, or other structures. Although they seem undifferentiated in the larval stages, the cells of the different discs are already destined to become a particular body part. Scientists can transplant a wing imaginal disc from a larva into the abdomen of an adult fly, leaving it there for months, and if it is transplanted back into a larva, it will still form wing tissue during pupation and morphogenesis.

A variety of experiments have shown that both the size of imaginal discs and the organs they form are very tightly controlled. When researchers transplant the wing imaginal disc from an early fly larva to a later one or vice versa, the wing still reaches normal size despite having different growing times. If researchers kill a portion of the imaginal disc cells with radiation or other techniques, the insect can boost cell division and still form a normal-size adult. If a fly receives just a fragment of a disc as a transplant, the animal won't move to the next stage of development until the disc has reached the correct size—pausing overall development to allow the disc to catch up. The transplanted disc "will know what size it should be," says developmental biologist Savraj Grewal of the University of Calgary in Canada.

Scientists can also change the rate at which imaginal disc cells divide, prompting either too many or not enough cells to form, but the cell size adjusts so that organ size remains the same. "The organ isn't counting cell divisions. It's measuring something about dimension," Grewal says. "What does the organ sense? I think the answer is still unclear."

There are a few clues, however. A protein called Hippo is part of a cascade of signals that plays a role in the growth of insect imaginal discs and some mammalian organs. (The name comes from the appearance of flies that lack the gene for Hippo in their eye imaginal discs; their overgrown heads have a wrinkled surface that resembles a hippopotamus hide.) Disruptions in the Hippo signaling pathway can lead to overgrowth, suggesting that it plays some role in controlling size. But recent studies suggest that it probably isn't the whole answer, says developmental biologist Georg Halder of the KU Leuven in Belgium.

Another set of genes that help with size sensing are ones that produce proteins called morphogens. These molecules originate from a single source in an embryo and diffuse across cells. They are best known for helping determine patterns during development. But some also influence the size of organs, tissues, and limbs. One theory is that when a morphogen gradient is steep—that is, there is a large difference in its concentration from one cell to another—then cells continue to divide. As cells divide, the gradient becomes more gradual. Once it has flattened out to a particular level, cells stop dividing.

There is also evidence that a cell's sense of which direction is up—called planar cell polarity—helps control growth. When certain genes involved in determining cell polarity are disrupted or missing, body parts tend to grow larger, suggesting that they can't sense when they should stop. "It's no coincidence that some of these polarity genes may act as tumor suppressors," Grewal says, and are suspected of playing a role in some cancers.

Many researchers suspect that a developing organ somehow senses the mechanical forces on its growing and dividing cells. One theory is that relative crowding and stretching of cells helps determine whether a cell continues to divide or stops.

The size of an organ depends not only on how many cells it has, but also how big those cells are. Some developing organs—plant leaves, for example, and fruit fly wings—can compensate when fewer cells are available by making the individual cells larger. How a leaf knows when to expand its cells is also unclear, says Hirokazu Tsukaya, a developmental biologist at the University of Tokyo who was among the first to characterize the phenomenon in leaves. He and his team have evidence that some sort of cell-to-cell communication drives the process. Here, too, the evidence suggests that a plant doesn't count cells but can somehow assess the overall size of a leaf, says plant biologist Beth Krizek of the University of South Carolina in Columbia. "But the mechanism of how that works is another mystery."

The size of tissues, and ultimately an overall organism, also clearly depends on signals from the environment, which researchers call extrinsic factors. Those size control systems are connected to, but different from, the intrinsic systems that help ensure an organism is correctly proportioned. In plants, growth can be especially sensitive to such outside factors, Krizek notes, because they can't move. Plants growing in shade, for example, concentrate on stem growth—to reach the sun—instead of leaf development. In animals, the amount of nutrition available can strongly influence the final size of some organs. One dramatic instance is the horn on a rhinoceros beetle. The horn is a sexually selected trait; males with bigger horns get access to more females. Recent studies have shown that the size of the horn is particularly sensitive to insulin signaling, which is related to the beetle's nutrition. That, in turn, signals the animal's overall fitness (Science, 27 July 2012, p. 408).

The problem of size control is still a fundamental one for developmental biologists, says Peter Lawrence of the University of Cambridge in the United Kingdom. Together with shape, size "is the material that evolution largely works on." But the field is still mostly in the dark. Despite hundreds of papers on what happens when the Hippo signaling pathway is interrupted, Lawrence notes, what scientists really need to understand is what it does when it is working properly. "That is not something we know."

6. Mysteries of Development

# Why Do So Many Neurons Commit Suicide During Brain Development?

1. Emily Underwood

Scientists have identified at least two, and possibly three, waves of neuronal cell death in the embryonic mammalian brain during development.

With a few notable exceptions, the roughly 100 billion neurons we have at birth are the only ones we'll ever have. Unlike skin and immune cells, which continuously self-renew, once a neuron has differentiated from its parent stem cell it will never divide again. Given this finite supply, why do so many neurons—more than half in some brain regions—kill themselves during embryonic brain development?

For roughly 50 years, many scientists focused on a single explanation for this rampant cellular suicide. Their hypothesis was rooted in research on the peripheral nervous system, which connects the nerves of the brain and spinal cord to limbs, organs, and sensory systems. To survive in the developing brain, researchers thought, neurons must compete for limited quantities of a chemical "trophic" factor released by the targets they aim to innervate. Without this signal, the cells self-destruct in a process known as programmed cell death, or apoptosis. Called the neurotrophic hypothesis, the concept neatly explained how an overabundance of neurons could attach where needed, or be culled.

"We were all carried away by this observation," says neuroscientist Yves-Alain Barde of the University of Basel in Switzerland. Inspired by the discovery of nerve growth factor in the 1950s, a protein essential to the growth and survival of sensory and motor neurons in the peripheral nervous system, Barde hunted for a single "survival" molecule for neurons in the brain. Although he hoped that his discovery in the 1980s of brain-derived neurotrophic factor would be that molecule, "this turned out not to be the case," he says. The protein does promote neuron survival in some parts of the brain, but it is more widely involved in stimulating their growth and shaping their connections.

Today, researchers recognize that the neurotrophic hypothesis alone cannot explain why so many cells die in the brain. "There's a complexity that we didn't appreciate," says Kevin Roth, a neuroscientist at the University of Alabama, Birmingham, School of Medicine. From the moment a neuron is born, he says, the cell is influenced not just by trophic factors, but also by a barrage of environmental and genetic cues that will ultimately determine whether it ever becomes a mature neuron.

Thanks to the invention of antibodies that can detect neurons as they self-destruct, scientists have identified at least two, and possibly three, waves of neuronal cell death in the embryonic mammalian brain during development, each tightly regulated by different enzymatic pathways. The first wave strikes down cells before they are fully differentiated neurons. Evidence suggests that this stage of cell death helps sculpt the size and shape of the nervous system, says neuroscientist Rae Nishi of the University of Vermont in Burlington.

Interfering with this process can have serious consequences. For example, by blocking the activity of caspases, a family of enzymes that aids in killing off young neurons, "you get really ugly, big brains," and the embryos soon die, Nishi says. Some caspases appear to target embryonic neurons with extra or missing chromosomes, suggesting that the programmed cell death culls abnormal cells, according to recent work from neuroscientist Jerold Chun at the University of California, San Diego.

A second wave of cell suicide occurs after neurons have begun to differentiate and extend their axons to make contact with other cells. Although some researchers have tried to "shoehorn" the die-off at this later stage into the neurotrophic hypothesis, Nishi says, she believes her group's work on neurons that innervate eye muscles suggests that lack of a survival molecule is not what triggers death at this time. Supporting this idea are new studies on cells in the cerebral cortex called inhibitory interneurons. Derek Southwell, a neuroscientist at Stanford University in Palo Alto, California, and colleagues transplanted a group of developing mouse interneurons into tissue where similar cells had already established themselves. In mice, 40% of inhibitory interneurons normally die during development after they migrate out of their birthplace in the forebrain to the cortex. Precisely 40% of the transplanted cells also died, suggesting that they acted of their own accord and not in response to their environment, Southwell says. "It seems there is some kind of developmental clock that times this decision about survival versus death," he concludes.

This strictly programmed cell death could prevent abnormal cells from being cancerous or remove cells that served some transient role. The cells might also require direct stimulation from other cells to survive and die off if they are isolated, Southwell suggests. "The truth of the matter is that we have no idea."

Such massive neuronal suicide could also have no purpose. In some cases, "blocking cell death doesn't have any dramatic consequences," Barde says. In 2006, for example, neuroscientist Ronald Oppenheim at the Wake Forest School of Medicine in Winston-Salem, North Carolina, was chagrined at the result of creating mice with absolutely no programmed cell death after neuronal differentiation. (He and his colleagues blocked the activity of a gene necessary for apoptosis.) "The most striking thing was that despite having all these tens of thousands of excess neurons throughout the nervous system, [the mice] seemed really quite normal," he says. "That was an embarrassing revelation. I thought, why have I been spending most of my career studying this?"

Although the excess cells look like motor neurons and have axons, they don't work. "They just hang around," Oppenheim says. "You might call them undead." He suggests that the developing nervous system compensates for the extra cells by simply not linking them in to neural circuits. Another possibility is that the effects of excess cells are too subtle to easily detect, Nishi says, adding that similar studies that have prevented normal cell death during the brain's development in mice have uncovered heightened anxiety and defensiveness.

Understanding the brain's profligate ways has become paramount. Overturning conventional wisdom, scientists now know that adults generate new neurons throughout their lives in a few select brain regions, such as the hippocampus. Many of these newcomers have short lifespans. "Of the immature neurons born in the hippocampus [during adulthood], the vast majority die before they're ever integrated into any kind of circuitry," Roth says. "Any time you have neurogenesis, the counter side is cell death." For now, however, why these processes are so deeply intertwined remains a mystery.

7. Mysteries of Development

# How Do Microbes Shape Animal Development?

1. Elizabeth Pennisi

There is a growing realization that microbes and their genes are partners in each animal's journey from egg to adult.

As the other mysteries in this package attest, developmental biologists have long had their hands full trying to fathom the transformation of a single cell into a full-fledged adult animal. They have concerned themselves primarily with deciphering the internal, genetically guided programs that take each species through specific stages to come out the right size and shape with a predetermined number of limbs, fins, eyes, and noses. But they were missing an elephant in the room, says Margaret McFall-Ngai, a developmental biologist at the University of Wisconsin, Madison: The world of microbes that live in, on, and around every animal.

The more she and other scientists get to know this world, the more they realize how big an influence microbes have on all aspects of animal and plant life—and not just as infectious pathogens. Take McFall-Ngai's research focus, the Hawaiian bobtail squid Euprymna scolopes. A nighttime hunter, it has evolved a way to acquire the bioluminescing bacterium Vibrio fischeri from surrounding seawater to light its underside so that predators below don't see its shadow in the moonlight. Squid embryos temporarily develop a mucus-laden ciliated patch inside the body cavity, where Vibrio selectively accumulate and eventually migrate into crypts destined to become the squid's so-called light organ. The presence of the bacteria affects squid gene activity, causing the ciliated patch to disintegrate and the light organ to differentiate. If there are no bacteria, the light organ fails to fully develop.

This example of Vibrios as midwives for the formation of an animal organ raises the provocative and, until recently, largely unaddressed question: How much do microbes shape normal development?

Animals and plants have always shared space with bacteria, fungi, viruses, and other microbes, coevolving through many millennia. In the mid-1800s, however, scientists came to view microbes primarily as enemies and fought hard with antibiotics, vaccines, and good hygiene to get the best of them. But the microscopic world is so intertwined with macroscopic life that the idea that each multicellular animal exists as a separate individual defined by its genome is falling by the wayside. There is a growing realization that microbes and their genes are partners in each animal's journey from egg through adulthood. "What we understand to be the 'individual' develops as a consortium of animal cells and microbes," says Scott Gilbert, a developmental biologist from Swarthmore College in Pennsylvania.

"Microbes came before us, so all development that takes place in all organisms has basically been taking place in the presence of the microbiota," adds Sven Pettersson of the Karolinska Institute in Stockholm.

The evidence for coevolution in developmental processes is coming from far corners of the animal kingdom. Whereas marine biologists once thought that drifting larvae of coral, snails, and other oceangoing invertebrates randomly settled down to become adults, they now know that many respond to cues from bacterial biofilms to pick their new homes. And while many animals develop in wombs or eggs apparently free of microbes, they may still rely on microbes to set in motion or complete certain aspects of postnatal development. Like McFall-Ngai's squid, mammals acquire microbial partners after birth and seem to have evolved strategies to encourage the right species to settle in specific places. Human milk, for example, contains complex sugars that infants cannot digest but which promote the growth of intestinal bifidobacteria.

But what do these microbial partners do? Germfree mice have finally allowed researchers to begin addressing this question. These are mice that lack the usual complement of gut bacteria because they are bred and raised in sterile environments and eat sterilized food. Studies of such mice make an increasingly strong case that bifidobacteria and other gut bacteria guide the postnatal maturation of the intestinal and immune systems, and even parts of the brain, in mammals. The microbes turn on mammalian genes important for cellular differentiation and produce metabolic products that may also affect development. Gut-associated lymphoid tissue and the capillary beds of the villi of the intestine fail to adequately develop in germfree mice, for example. With respect to the immune system, mouse studies also show that a polysaccharide produced by the symbiont Bacteroides fragilis helps establish the right balance between helper 1 and helper 2 T cells. B cells also need symbiotic bacteria to develop normally.

The evidence for a role for symbionts in the postnatal developing brain is more preliminary but nonetheless intriguing. More and more connections are being found between the gut microbiota and behavior (Science, 12 October 2012, p. 198). In 2011, Pettersson and his colleagues tested anxiety levels and locomotor activity in germfree mice and found that the rodents are hyperactive and have a decreased level of anxiety compared with mice with a healthy microbiota. There were also differences in the activity of genes associated with motor activity and anxiety. There seems to be a window of opportunity for the microbiota to influence behavior patterns: Colonizing germfree mice with normal mouse microbes negated these differences in young, but not older, mice, they reported.

Some work suggests that gut microbes influence behavior through the vagus nerve, which connects the brain with the digestive system, but Pettersson and others suspect a role for blood-borne bacterial products as well. These products, which make up 10% or more of the metabolites in blood, may extend the reach of the gut microbiota throughout the body.

That realization may mean that prenatal development in mammals isn't as free from microbial influence as everyone has thought. In mammals, the developing fetus is virtually bacteria-free; hence, researchers have focused on finding a role for bacteria in development after birth. Yet blood-borne metabolites from a mother's gut germs could exert an effect on a growing fetus. "That was one of the assumptions, that pregnancy did not involve microbes," Gilbert says. "But it probably does."

As such assumptions are overturned, researchers are addressing new issues. What is the molecular dialogue that enables the microbial world to influence development? How did that dialogue evolve and how often is it a friendly one? "The big questions are now exposed," says Michael Hadfield, a developmental biologist at the University of Hawaii, Manoa. "After all the years we tended to ignore the bacteria, most people who are studying development should be looking for where the bacteria are and what roles they are playing."

8. Mysteries of Development

# How Does Fetal Environment Influence Later Health?

1. Jennifer Couzin-Frankel

There's broad agreement that the fetal world, the most rapid period of human growth and development, shapes one's risk of future disease, although how much influence it has remains uncertain.

Parents pore over their newborn's face, drinking in the fuzz of her eyebrows, the shape of the chin, searching for themselves in her smile. But they're not thinking about what they can't see, and what ultimately matters more: the heft of her heart, the hormones churning from the liver, all those invisible features that influence her health into adulthood.

While their baby's biology of course reflects a mingling of the mother's and father's DNA, there's more to her than that. In a peculiar way, all newborns are "an expression of the mother," in the words of David Barker, a physician and epidemiologist at the University of Southampton in the United Kingdom. He believes that people are shaped, inside and out, by the maternal environment that sustained them before they were born.

In the late 1980s, Barker scrutinized thousands of birth and death certificates of people from Hertfordshire, U.K., and concluded that those whose birth weight fell on the low end of normal were much more likely to die of heart disease as adults. Since then, Barker has promulgated his theory that maternal environment controls a baby's destiny in more ways than we yet understand.

These days, there's broad agreement that the fetal world, the most rapid period of human growth and development, shapes one's risk of future disease, although how much influence it has remains uncertain. A key missing link is in the mechanism. What switches in the fetus, or the placenta that nourishes it, are flipped by a mother's diet or stress levels? In other words, how does fetal environment mold development?

Those exploring this fundamental mystery have at least one intriguing discovery to follow up. No matter what the stressor on the fetus, studies of people and animals suggest that the output is similar: a higher risk of type 2 diabetes, obesity, heart disease, insulin resistance, and high blood pressure. In rodents, "anything that could be a nutritional stressor seems to have the same effect," says Simon Langley-Evans of the University of Nottingham in the United Kingdom, suggesting that the fetus is implementing a universal response to stress, perhaps to ensure its survival.

The early focus of the field that Barker spawned was on birth weight, a crude reflection of a fetus's surroundings: Smaller babies tended to reflect poorly nourished or highly stressed mothers. But what a mother eats when she's pregnant is only a small part of the fetal environment, Barker notes. "The mother's body is the product of her lifetime nutrition," he says—and even her own mother's nutrition, too, because most or all of her eggs are formed before birth.

Scientists are now striving for greater sophistication in exploring the black box of the womb. Animal studies have found that without good nutrient flow across the placenta, the offspring responds "by building its organs on the cheap," says Kent Thornburg, a cardiac physiologist at Oregon Health & Science University in Portland. Hearts have fewer muscle cells. Kidneys have fewer nephrons for filtering urine. There's less skeletal muscle in limbs and fewer insulin-producing cells in the pancreas.

Peeling back the layers, scientists are also finding differences in DNA patterns in the offspring, depending on whether their mothers were properly fed or malnourished. One long-running effort examines men and women who developed in utero during the Dutch Hunger Winter of 1944 to 1945, when the Germans cut off food and fuel shipments to part of the Netherlands. A birthday soon after was correlated with more obesity and impaired glucose metabolism in adulthood, along with higher rates of other health issues. In 2008, a group at Columbia University and Leiden University Medical Center added a genetic twist to that well-documented story. They reported that almost 60 years after the famine, those born at the time had different patterns of methylation, a chemical coating of DNA that influences gene expression, in the gene IGF2 as compared with their siblings who arrived in flusher times. The researchers also found that as adults, men had more differences in methylation than women born at the same time. They are continuing to explore methylation patterns on a genomewide scale among their cohort.

One problem with this work is that no one knows for sure whether the DNA changes occurred in utero in response to the famine, or came about later in life for entirely different reasons. Nor do scientists yet know whether these genetic changes, which are often modest, play a role in disease susceptibility. "It's so damn difficult" to do this research, says Bastiaan T. Heijmans of Leiden University Medical Center, who led these methylation studies of the Dutch Hunger Winter cohort along with his Columbia colleague, Lambert H. Lumey. "There's some quite compelling evidence that indeed this relationship is there" between the fetal environment, the DNA changes, and later health problems. But "it's hard for me to put my finger on" exactly what's going on.

Animal work can help clear up the confusion. And it, too, is identifying striking sex differences in how fetuses react to their surroundings. Ten years ago, Lubo Zhang of Loma Linda University School of Medicine in California began depriving pregnant rats of oxygen, mimicking the effects of a mom-to-be with heart disease, or women whose placentas are poorly formed. Then he studied the hearts of the offspring when they reached adulthood.

The organs, Zhang found, functioned normally. That is, until he induced stress in the animals that mimicked a heart attack. The males lost far more heart muscle tissue than females or than animals whose gestation had been healthy. "They become much more vulnerable to the second hit," he says. "If the heart is not stressed later in life, [the animals] cannot tell the difference."

Over the ensuing years, Zhang traced this effect to dampened expression in a gene called Protein Kinase C epsilon. Protecting fetuses from low oxygen, for example with a compound called N-acetylcysteine, kept this gene's activity up during development and hearts healthy long-term. He hypothesizes that extra estrogen in the placentas of female animals protects them.

Then there are the myriad studies suggesting that pregnant women (or pregnant rodents) who suffer from common infections like the flu or a days-long fever are more likely to have offspring who develop autism or schizophrenia. Here, too, the findings are still tenuous, and researchers are only beginning to address how a woman's immune system battling infection can influence the developing nervous system in her womb.

One underexplored piece of gestation is the placenta. Its morphology varies tremendously—perhaps related to a mother's body composition and diet—and studies have found that the state of the placenta at a baby's birth can predict how the child fares later on. Barker, Thornburg, and others have probed this connection via the Helsinki Birth Cohort Study of more than 20,000 people born in the 1930s and '40s. The hospitals kept detailed measurements of each baby's placentas, and the researchers have linked placental measurements to later adult health, everything from sudden cardiac death to lung cancer to insulin resistance. As with so many fetal environment studies, though, the choreography—the pattern of dance steps that occur between fetus and placenta—is largely unknown.

Solving these mysteries is daunting. "We try to link time points that are so far apart," Heijmans says. "There's no study that goes from preconception to 100 years of age." While "a good part of healthy aging starts in the womb," Heijmans believes, it's just the beginning to a hopefully long life that will mold health and disease.

9. Mysteries of Development

# Under Development

1. Gretchen Vogel

Much of what we know about the journey from single cell to mature organism involves what happens when things go awry. Here are five more mysteries of what happens when genes are working as they should.

The journey from single cell to mature organism is full of intrigue. Far too much of what we know about development involves what happens when things go awry, says Peter Lawrence of the University of Cambridge in the United Kingdom. "If a mutant gene causes an organism's head to fall off, the conclusion is that the gene's function is to hold the head on," he says. "People have applied this logic, inappropriately, to complex phenomena like the building of an organism." The focus on mutations, he says, has distracted the field from some of the most important questions in development, which require understanding what genes do when they are working as they should. Here are five more mysteries of development.

The shape of things to come. Despite exciting progress reported in this issue (see p. 1183), how cells use genetic instructions to form the shapes that organisms ultimately take is a conundrum. "The shape of your nose? That's all written very precisely somewhere in some form," Lawrence says. "We have no idea where."

Not carbon copies. Although they share the same genome, identical twins are different—sometimes subtly, sometimes dramatically. They show how chance events can influence developing organisms, but many questions remain about just how much of development is due to chance.

Millennial naps. Researchers recently coaxed seeds to sprout after being buried in frozen tundra for thousands of years. How do seeds remain in a state of suspended animation, waiting for the right moment to start putting down roots and pushing up shoots?

Pick your progeny. How do stem cells—which can both replicate themselves and give rise to other cell types—know when to switch from one kind of daughter cell to another?

New parts from old. During evolution, new structures such as turtle shells or bat wings arise through a process that repurposes existing parts. Tracing the genetic changes that lead to new structures as species evolve is a passion of evolutionary developmental biologists; advances in genomics may help solve such mysteries.

10. Bioelectronics

# The Cyborg Era Begins

1. Robert F. Service

Advances in flexible electronics now make it possible to integrate circuits with tissues.

John Rogers doesn't look like a cyborg yet, but his transformation has begun. As he delivered a talk at a recent conference in San Francisco, Rogers, a materials scientist at the University of Illinois, Urbana-Champaign, picked up a penlike microscope connected to the projector that beamed the PowerPoint slides on his computer for all in the audience to see. As Rogers pressed the pen-scope against his forearm, viewers got a close-up view of the craggy hills and valleys of his skin, as well as an array of squiggly gold lines and square pads. The lines and pads, it turns out, were components of arrays of circuits—not the Intel or ARM variety found in your laptop or cell phone, but a postage stamp–sized collection of flexible, stretchable, and nearly transparent devices that molded perfectly to the contours of Rogers' skin.

That intimate contact, Rogers explained, will allow his team to use flexible circuitry to monitor body temperature, heart rate, and blood pressure and wirelessly transmit the data to a nearby computer. Using similar arrays, Rogers's team has also been able to track arm motion, allowing researchers to control a toy helicopter's flight path with a wave of the arm. Through a startup company that he founded called MC10, Rogers has teamed up with NBA and NFL stars such as Grant Hill and Matt Hasselbeck to use the technology to monitor head impacts during sports. Working with the stars "is pretty cool," Rogers says. "It gives you a lot of credibility with your 10-year-old son."

Fanciful as such technology sounds, it's just the beginning. Additional devices shown by Rogers and others at the Materials Research Society (MRS) meeting went even further. Cling wrap–like circuitry draped over the hearts of test animals can not only independently track the activity of each of the heart's four chambers, but it can also emit pulses of heat that kill tiny patches of tissue that initiate potentially deadly arrhythmias. Other arrays penetrate brain tissue to monitor the abnormal nerve firing patterns in epilepsy or induce gene expression in the brain tissue of mice and monitor the results to study developmental biology. One team has even made a 3D printed bionic ear complete with cartilage cells and wiring able to tune in to both Beethoven's "Für Elise" and ultrasonic bleats that humans cannot hear.

Meet your future self. The beginnings of a cyborg world have arrived. These early prototypes are pale shadows of the Hollywood versions, such as the humanoid Cylons in Battlestar Galactica or the Borg in Star Trek who claim that resistance to incorporation into their collective is futile. Nevertheless, research progress is real, as a mix of biologists, materials scientists, and nanotechnology experts are chipping away at a host of challenges. "I see it as building a seamless interface between cells, tissues, and electronics," says Aleksandr Noy, a bionanoelectronics expert at Lawrence Livermore National Laboratory and the University of California, Merced. For now, most of these efforts focus on providing better health care and quality of life for patients. But over time, expect devices that will make us better athletes and soldiers, or even improve our complexion.

"A few years ago these things were science fiction. Now we are seeing the emergence of real devices and applications," Noy says. And fast, says Zhenan Bao, an organic electronics expert at Stanford University in California: "The competition is furious."

## Stretching the limits

The idea of fusing man and machine has long tantalized humanity. Over the past century, Rogers points out, researchers have pioneered myriad efforts to use electronics to measure biological activity and sometimes even alter it. They tailored metal electrodes that could be taped to the skin for use in electrocardiograms. They devised brain stimulators that can be inserted deep within brain tissue to disrupt the neural firing patterns that cause debilitating tremors in patients with Parkinson's disease. And they created cochlear implants capable of converting sound to electrical impulses that can be registered by the inner ear.

The early technologies were crude: rigid devices that were strapped and glued to the skin or stabbed through soft tissue. The latest iterations are more about tailoring electronics to mimic the body's pliability. Efforts range from the macroscale of things we can see to the microscopic scale at which electronics are being entwined with individual cells.

At the upper end of the scale, Michael McAlpine, a mechanical engineer at Princeton University, and colleagues reported in the 1 May issue of Nano Letters that they've made the first 3D printed functional organ: a bionic ear that hears acoustic sound and ultrasound. "We're trying to see if one could introduce augmented functionality that a human wouldn't ordinarily have," McAlpine says.

Three-dimensional printers work by using a computer-driven laser printer to build up layers of material-based inks, usually made from plastics. McAlpine's team started with three different inks: one made from silicone; another with silicone infused with silver nanoparticles; and a third with chondrocytes, cells that produce cartilage, along with a gel to promote their growth. Numerous groups have used 3D printing to make tissues, but they have typically printed only scaffolding material and cells. McAlpine's team added a level of sophistication to the technology. The researchers printed out a metal coil in the center of the engineered ear that serves as an antenna capable of picking up acoustic signals and converting them to electrical pulses for the inner ear, à la a conventional cochlear implant. The antenna can also detect ultrasonic waves that dogs and other animals can hear but humans cannot.

Another macroscopic project is electronic skin, complete with tactile, temperature, and even chemical sensors. Such skin could potentially be integrated into prosthetic limbs, enabling users to feel and touch their world again, and perhaps give robots a new sense of their surroundings. For electronic skin to work, it must be soft, flexible, and stretchable, much like our own. That rules out conventional computer electronics made from rigid glass and ceramic chips.

But such skin is around the corner thanks to progress in organic electronics. Nearly all organic solids are insulators: They don't readily conduct electricity. But in the 1970s and 1980s, researchers discovered how to tweak the structures of some organic compounds to make them metal-like conductors or semiconductors, with the ability to switch a current on and off—a property critical to transistors and other devices. That advance opened the door to creating electronics on soft, flexible substrates. By the 2000s, researchers had honed their skills to make arrays of devices and pattern them cheaply. Compared with top-shelf electronics in computers and cell phones, flexible electronics remained big and slow. But they could now go places that rigid silicon could not.

In the past few years, numerous groups have unveiled flexible and stretchable arrays of touch and temperature sensors. For example, 2 years ago Bao and her colleagues at Stanford made an array of flexible organic pressure sensors so sensitive that it could detect the weight of a butterfly sitting on top. More recently, at the MRS meeting, Bao reported how she and her colleagues had used a miniature array, roughly 144 square millimeters in area, to detect heartbeat and blood pressure from a simple watch-style wristband. Bulky wristwatch heart rate monitors exist, but the Stanford team's devices are paper-thin. "Human skin is a great inspiration," says Bao, whose group has also produced flexible chemical sensors and devices in which metal nanoparticles can move to fill in cracks so that damaged devices can heal themselves.

According to the online rumor mill that follows everything that the electronics giant Apple has in the works, the company is testing the use of flexible electronics in a sleek iWatch that will have a screen for surfing the web, as well as monitoring time, temperature, and fitness information such as how many calories a jogger wearing the device is burning up. Apple isn't alone. MC10 and fitness giant Reebok have developed prototypes of wearable sensors to monitor concussion risk in athletes playing contact sports like football or hockey. If the device senses strong enough head impacts—as determined by measurements of rotational acceleration, multidirectional acceleration, and the location and duration of the blow—it lights up a panel of light-emitting diodes (LEDs) that alerts the coach to take the player out of the game.

Other researchers are shrinking devices to pack more into a patch. One technology at this frontier is touch sensors. Most touch sensors work by spotting how pressure changes the electrical resistance of current flowing through a device array. Chemist Zhong Lin Wang of the Georgia Institute of Technology in Atlanta and his colleagues have come up with an alternative based on piezotronics, in which increasing the strain on a material changes its polarization, or the distribution of positive and negative charges, which can be read out as a signal. Unlike the conventional transistor approach, Wang notes, piezotronics enable researchers to fashion tiny devices, making it possible to create arrays with ultrahigh resolution. In the 24 May issue of Science (p. 952), the group reported packing piezotronic transistors at the density of 8464 devices per 1 centimeter square—thousands of times denser than tactile sensors in human skin. Such arrays, Wang says, could eventually be useful in providing high-resolution touch sensing to prosthetic limbs, or giving robots a tactile sense sharp enough to identify what they're touching, or making signature readers capable of sensing not only the swoops of a handwritten signature but characteristic changes in pressure as well.

Flexible sensors are going way past skin deep. Rogers's team reported last year in the Proceedings of the National Academy of Sciences that ultraflexible circuitry could cling to the surface of beating hearts of pigs and rabbits and, at each of more than a dozen points in the array, continuously register the electric signals that fire muscle cells in the beating heart. This allowed them to image waves of muscle fiber contraction as blood is pumped throughout different chambers. The fine detail also makes it possible to map patches of heart tissue that misfire during arrhythmia, a condition that affects up to 5% of people in the United States.

Rogers and his colleagues also integrated temperature sensors and tiny heating pads into their array. By turning on the pads—emitting heat instead of measuring it—they ablated tiny tissue patches in their test animals, opening the door to using similar arrays to cure arrhythmia.

## Going deep

Perhaps the boldest direction in bioelectronics is the emerging effort of marrying tissue and electronics at the cellular level. At Harvard University, chemist Charles Lieber and his colleagues have spent much of the past 2 decades pioneering efforts to grow ultrathin nanowires from the atomic scale up and to design them to work as transistors and other electronic devices. Nanoscale devices are the right size to monitor and influence biology inside cells. "It's really the natural length scale for electrical interface," Lieber says. Ion channels in neurons are less than 10 nanometers in width, synaptic connections between nerves are less than 100 nanometers across, and neurons themselves are on the order of a micrometer. Devices at those scales could be revolutionary.

Using nanowires, Lieber's team has made nanoscale computer memories, LEDs, and photovoltaics. In a series of recent papers, Lieber and his colleagues used their devices to track nerve firing. And they've placed multiple transistor-based recorders on nanowire probes that can be inserted inside neurons, making it possible for the first time to track neural signals as they traverse the interior of a cell. Patch clamp probes have made it possible to monitor the firing of individual cells since the late 1970s, but that approach can't track action potentials inside a cell. The new nanowire approach "is really the first fundamentally new recording method since the patch clamp," Lieber says.

Another new device in the nano tool chest is one that uses pulses of light to control gene expression in mice. Optogenetic tools of late have become a valuable way for biologists to initiate the expression of target genes in lab animals and track the effect. To use them, however, researchers typically must tether animals to complex electronic gear that distorts their natural behavior. In the 12 April issue of Science, Rogers and his colleagues described patterning nanoscale LEDs on the end of a flexible nanoscale filament (p. 211). These nano LEDs were then injected deep within the brain tissue of mice engineered to turn on particular genes in response to light. The nanoribbons were designed to pick up nearby radio waves and convert them to electricity. By placing a radio source next to the heads of the mice, the researchers turned on the LEDs, which, in turn, triggered gene expression. The new technique, Rogers says, foreshadows applications in which self-powered bioelectronics embedded in a variety of organs will regulate their function.

Some of the latest work by Lieber's group makes that day seem close at hand. As described in the November 2012 issue of Nature Materials, his team created a 3D mesh of silicon nanowires with built-in transistors. They used the mesh as scaffold for growing tissue, either heart muscle cells or neurons. The transistor array could monitor the electrical activity of the growing tissues and track their response to drugs such as noradrenaline, which stimulates cardiac cell contraction.

Such devices are only the beginning of a brave new world of bioelectronics. Better hope that it is more benign than Star Trek's soulless collective, because make no mistake: With so much to gain, we will be assimilated.