Modeling the Genes for Development

Caltech biologists create the first predictive computational model of gene networks that control the development of sea-urchin embryos

PASADENA, Calif.—As an animal develops from an embryo, its cells take diverse paths, eventually forming different body parts—muscles, bones, heart. In order for each cell to know what to do during development, it follows a genetic blueprint, which consists of complex webs of interacting genes called gene regulatory networks.

Biologists at the California Institute of Technology (Caltech) have spent the last decade or so detailing how these gene networks control development in sea-urchin embryos. Now, for the first time, they have built a computational model of one of these networks.

This model, the scientists say, does a remarkably good job of calculating what these networks do to control the fates of different cells in the early stages of sea-urchin development—confirming that the interactions among a few dozen genes suffice to tell an embryo how to start the development of different body parts in their respective spatial locations. The model is also a powerful tool for understanding gene regulatory networks in a way not previously possible, allowing scientists to better study the genetic bases of both development and evolution.

"We have never had the opportunity to explore the significance of these networks before," says Eric Davidson, the Norman Chandler Professor of Cell Biology at Caltech. "The results are amazing to us."

The researchers described their computer model in a paper in the Proceedings of the National Academy of Sciences that appeared as an advance online publication on August 27.

The model encompasses the gene regulatory network that controls the first 30 hours of the development of endomesoderm cells, which eventually form the embryo's gut, skeleton, muscles, and immune system. This network—so far the most extensively analyzed developmental gene regulatory network of any animal organism—consists of about 50 regulatory genes that turn one another on and off.

To create the model, the researchers distilled everything they knew about the network into a series of logical statements that a computer could understand. "We translated all of our biological knowledge into very simple Boolean statements," explains Isabelle Peter, a senior research fellow and the first author of the paper. In other words, the researchers represented the network as a series of if-then statements that determine whether certain genes in different cells are on or off (i.e., if gene A is on, then genes B and C will turn off).

By computing the results of each sequence hour by hour, the model determines when and where in the embryo each gene is on and off. Comparing the computed results with experiments, the researchers found that the model reproduced the data almost exactly. "It works surprisingly well," Peter says.

Some details about the network may still be uncovered, the researchers say, but the fact that the model mirrors a real embryo so well shows that biologists have indeed identified almost all of the genes that are necessary to control these particular developmental processes. The model is accurate enough that the researchers can tweak specific parts—for example, suppress a particular gene—and get computed results that match those of previous experiments.

Allowing biologists to do these kinds of virtual experiments is precisely how computer models can be powerful tools, Peter says. Gene regulatory networks are so complex that it is almost impossible for a person to fully understand the role of each gene without the help of a computational model, which can reveal how the networks function in unprecedented detail.

Studying gene regulatory networks with models may also offer new insights into the evolutionary origins of species. By comparing the gene regulatory networks of different species, biologists can probe how they branched off from common ancestors at the genetic level.

So far, the researchers have only modeled one gene regulatory network, but their goal is to model the networks responsible for every part of a sea-urchin embryo, to build a model that covers not just the first 30 hours of a sea urchin's life but its entire embryonic development. Now that this modeling approach has been proven effective, Davidson says, creating a complete model is just a matter of time, effort, and resources. 

The title of the PNAS paper is "Predictive computation of genomic logic processing functions in embryonic development." In addition to Peter and Davidson, the other author on the PNAS paper is Emmanuel Faure, a former Caltech postdoctoral scholar who is now at the École Polytechnique in France. This work was supported by the National Institute of Child Health and Human Development and the National Institute of General Medical Sciences.

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Caltech Researchers Find Evidence of Link between Immune Irregularities and Autism

PASADENA, Calif.—Scientists at the California Institute of Technology (Caltech) pioneered the study of the link between irregularities in the immune system and neurodevelopmental disorders such as autism a decade ago. Since then, studies of postmortem brains and of individuals with autism, as well as epidemiological studies, have supported the correlation between alterations in the immune system and autism spectrum disorder.

What has remained unanswered, however, is whether the immune changes play a causative role in the development of the disease or are merely a side effect. Now a new Caltech study suggests that specific changes in an overactive immune system can indeed contribute to autism-like behaviors in mice, and that in some cases, this activation can be related to what a developing fetus experiences in the womb.

The results appear in a paper this week in the Proceedings of the National Academy of Sciences (PNAS).

"We have long suspected that the immune system plays a role in the development of autism spectrum disorder," says Paul Patterson, the Anne P. and Benjamin F. Biaggini Professor of Biological Sciences at Caltech, who led the work. "In our studies of a mouse model based on an environmental risk factor for autism, we find that the immune system of the mother is a key factor in the eventual abnormal behaviors in the offspring."

The first step in the work was establishing a mouse model that tied the autism-related behaviors together with immune changes. Several large epidemiological studies—including one that involved tracking the medical history of every person born in Denmark between 1980 and 2005—have found a correlation between viral infection during the first trimester of a mother's pregnancy and a higher risk for autism spectrum disorder in her child. To model this in mice, the researchers injected pregnant mothers with a viral mimic that triggered the same type of immune response a viral infection would.

"In mice, this single insult to the mother translates into autism-related behavioral abnormalities and neuropathologies in the offspring," says Elaine Hsiao, a graduate student in Patterson's lab and lead author of the PNAS paper. 

The team found that the offspring exhibit the core behavioral symptoms associated with autism spectrum disorder—repetitive or stereotyped behaviors, decreased social interactions, and impaired communication. In mice, this translates to such behaviors as compulsively burying marbles placed in their cage, excessively self grooming, choosing to spend time alone or with a toy rather than interacting with a new mouse, or vocalizing ultrasonically less often or in an altered way compared to typical mice. 

Next, the researchers characterized the immune system of the offspring of mothers that had been infected and found that the offspring display a number of immune changes. Some of those changes parallel those seen in people with autism, including decreased levels of regulatory T cells, which play a key role in suppressing the immune response. Taken together, the observed immune alterations add up to an immune system in overdrive—one that promotes inflammation.

"Remarkably, we saw these immune abnormalities in both young and adult offspring of immune-activated mothers," Hsiao says. "This tells us that a prenatal challenge can result in long-term consequences for health and development."

With the mouse model established, the group was then able to test whether the offspring's immune problems contribute to their autism-related behaviors. In the most revealing test of this hypothesis, the researchers were able to correct many of the autism-like behaviors in the offspring of immune-activated mothers by giving the offspring a bone-marrow transplant from typical mice. The normal stem cells in the transplanted bone marrow not only replenished the immune system of the host animals but altered their autism-like behavioral impairments. 

The researchers emphasize that because the work was conducted in mice, the results cannot be readily extrapolated to humans, and they certainly do not suggest that bone-marrow transplants should be considered as a treatment for autism. They also have yet to establish whether it was the infusion of stem cells or the bone-marrow transplant procedure itself—complete with irradiation—that corrected the behaviors.

However, Patterson says, the results do suggest that immune irregularities in children could be an important target for innovative immune manipulations in addressing the behaviors associated with autism spectrum disorder. By correcting these immune problems, he says, it might be possible to ameliorate some of the classic developmental delays seen in autism.

In future studies, the researchers plan to examine the effects of highly targeted anti-inflammatory treatments on mice that display autism-related behaviors and immune changes. They are also interested in considering the gastrointestinal (GI) bacteria, or microbiota, of such mice. Coauthor Sarkis Mazmanian, a professor of biology at Caltech, has shown that gut bacteria are intimately tied to the function of the immune system. He and Patterson are investigating whether changes to the microbiota of these mice might also influence their autism-related behaviors.

Along with Patterson, Hsiao, and Mazmanian, additional Caltech coauthors on the PNAS paper, "Modeling an autism risk factor in mice leads to permanent immune dysregulation," are Mazmanian lab manager Sara McBride and former graduate student Janet Chow. The work was supported by an Autism Speaks Weatherstone Fellowship, National Institutes of Health Graduate Training Grants, a Weston Havens Foundation grant, a Gregory O. and Jennifer W. Johnson Caltech Innovation Fellowship, a Caltech Innovation grant, and a Congressionally Directed Medical Research Program Idea Development Award. 

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Kimm Fesenmaier
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Autism and the Immune System
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Caltech Receives Gift from Sackler Foundation to Advance Biomedical Science Research

PASADENA, Calif.—The California Institute of Technology (Caltech) and UCLA have launched highly productive collaborations in cancer research and other areas of biomedicine in recent years, frequently through the Caltech lab of Nobel Laureate and President Emeritus David Baltimore. Now, an endowment established by the Raymond and Beverly Sackler Foundation will strengthen the Caltech-UCLA partnership and advance the Baltimore lab's interdisciplinary research into areas where mathematics and engineering converge with biology.

The Sackler endowment will support three primary areas: postdoctoral researchers working in areas that have a convergent theme; students pursuing a joint MD/PhD degree through the Caltech-UCLA Medical Scientist Training Program; and a joint seminar series between Caltech and UCLA emphasizing the interface of medicine, engineering, and the physical sciences.

"Caltech has a strong interdisciplinary focus that is fueling its outstanding achievements in biomedical research," says Caltech's president Jean-Lou Chameau. "The support from the Raymond and Beverly Sackler Foundation further enhances this research, which can lead to therapies for some of the world's most critical diseases."

The research supported by the endowment will encompass many of the numerous disciplines that have become intertwined with biology in recent years, such as computational mathematics and physics. There is also an important educational component, as the Sackler endowment will help train MD/PhD students and strengthen the link between Caltech's basic science expertise and UCLA's translational medicine focus.

"The Sackler gift is very important because it allows us to expand our research in a direction in which I very much want it to go, and at the same time it will support our very significant MD/PhD program," says Baltimore, the Robert Andrews Millikan Professor of Biology. "We are grateful to Raymond and Beverly Sackler for their generosity and for their foresight in recognizing the importance of our work."

Dr. Raymond Sackler is a physician, entrepreneur, and philanthropist who has supported numerous scientific and cultural initiatives throughout the world. With his late brothers, Arthur and Mortimer, he sponsored the Sackler Faculty of Medicine, and with his wife, Beverly, has sponsored the Raymond and Beverly Sackler Faculty of Exact Sciences, both at Tel Aviv University. David Baltimore and Caltech are one of 10 recipients of endowments from the foundation as part of a recent global program in support of convergent research.

"The purpose of this gift is to catalyze new convergent science investigations and to honor the important research that has been carried out by David Baltimore," says Raymond Sackler. "He made groundbreaking scientific accomplishments at a relatively early age, and now—working at the crossroads of several disciplines—he continues to make major discoveries in biomedical research. We hope that this new endowment will foster his research and Caltech's dynamic collaborations with UCLA."

Baltimore is best known for his work with viruses and the immune system, and he shared the Nobel Prize in Physiology or Medicine in 1975 for his discoveries concerning cancer-inducing viruses. Several years ago, he decided that his research should move toward translational applications, and in 2009 he became the director of a new effort with UCLA—the Joint Center for Translational Medicine—which supports numerous research projects with the potential for clinical applications.

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Michael Rogers
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Caltech Biologist Stephen Mayo Named Inaugural Bowes Division Chair

Bowes Foundation gift will seed innovative projects in the biological sciences

PASADENA, Calif.—Stephen L. Mayo, chair of the Division of Biology and Bren Professor of Biology and Chemistry at the California Institute of Technology (Caltech), has been named the William K. Bowes Jr. Foundation Division Chair. The William K. Bowes, Jr. Foundation, based in San Francisco, endowed the new division leadership chair with a $5 million gift, supplemented by an additional $2.5 million provided by the Gordon and Betty Moore Matching Program.

The new chair is named after the philanthropic foundation created by William K. Bowes, Jr., founding partner of U.S. Venture Partners in Silicon Valley.  For nearly 50 years, Bowes has helped launch numerous biotechnology and other high-tech companies, including Amgen, Applied Biosystems, and Sun Microsystems.

Unlike typical professorships, which fund salaries, the Bowes Foundation Chair—also called a leadership chair—is a permanent endowment that will provide funds that Mayo and future biology division chairs will use to support innovative research projects with potential for scientific and societal impact. It will also be used to support Caltech's education mission and outreach programs.

"The Bowes Chair is unprecedented at Caltech and will be a tremendous asset to advance our science and engineering research and teaching," says Caltech president Jean-Lou Chameau. "This is the first in what will be a series of leadership chairs providing high-leverage, unrestricted support that the faculty who head our divisions will use to seed the Institute's most ambitious educational and research ideas. We are extremely grateful for Bill Bowes's inspirational and generous support."

Bowes has been connected with Caltech for more than 30 years, most notably through the founding of Amgen, which included Caltech biologists Norman Davidson and Leroy Hood as two of its scientific founders; they were also original members of the company's scientific advisory board.  "I have known firsthand for years that Caltech faculty have the unique intellectual skills, imagination, and track record to propel promising entrepreneurial ventures," says Bowes. "With the Bowes Foundation Division Chair, I hope to give Caltech's extremely talented biological scientists the freedom to turn more of their ideas into reality. This is really a carefully considered investment in the future."

 "Bill Bowes's important gift will give Caltech's biology division critical unrestricted funding to foster valuable research, teaching, and outreach programs," says Mayo. "It is an honor to be named the first Bowes Foundation Division Chair, not only because this is such a groundbreaking initiative for Caltech, but also because Bill has been so instrumental in launching entrepreneurial ventures that have improved so many people's lives."

Bowes has had both a varied and an influential career. After service in the Army infantry in the South Pacific and Japan during and after World War II, Bowes received a BA in economics from Stanford and then an MBA from Harvard. He was an investment banker in San Francisco for nearly three decades before founding U.S. Venture Partners in 1981. He founded and served as the first chairman and treasurer of Amgen, which would become the world's largest biotech company.

U.S. Venture Partners has played a leading role in the development of the software, health-care, and e-commerce industries, building companies such as Sun Microsystems and Applied Biosystems. It has also been one of the leading investors in Caltech start-ups, including Axiom Microdevices, Cleave Biosciences, Contour Energy Systems, and Proteolix. (For more on these companies, see "A Snapshot of Start-ups.")

In recent years, Bowes has focused his energies on philanthropy, supporting nonprofit organizations in such areas as medical research, higher education, and the environment. He has also served on numerous committees and boards for a broad range of institutions, including the Environmental Defense Fund, the San Francisco Exploratorium, the Hoover Institution, and the San Francisco Conservatory of Music.

"Bill Bowes has not only been one of the nation's pioneers in venture capital, but he has also demonstrated the effectiveness of prudent investing in philanthropy," says Caltech senior trustee William Davidow, founding partner of Mohr Davidow Ventures, a venture-capital firm based in Menlo Park. "The early experimental research that Bill is supporting today at Caltech will become the innovative entrepreneurial enterprises of tomorrow."

Steve Mayo is a pioneer in protein-design technology, having been the first to design a protein on a computer and then build it in a lab. Working at the interface of theory, computation, and wet-laboratory experimentation, Mayo focuses on developing quantitative approaches to protein engineering, aiming to understand the physical and chemical determinants of protein structure, stability, and function.

His design approach has been incorporated in a suite of software programs called Comprehensive Protein Design Software, and has been applied to a variety of problems ranging from protein fold stabilization to enzyme design. The end goal, he says, is to create protein-based therapies against diseases, new ways of improving agricultural production, and other applications. Mayo has founded or cofounded several companies, including Molecular Simulations, Xencor, and Protabit.

Caltech has a distinguished history of discovery in the biological sciences, recognized by numerous Nobel Prizes. Over its more than 75 years, Caltech's Division of Biology has made many of the research advances that led to the biotechnology and genetic revolutions. In the past, Caltech's biologists made fundamental insights in cellular and molecular biology; today our investigators are both continuing to blaze that path of discovery and applying their knowledge to finding innovative solutions to cancer, AIDS, and other diseases.

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The Physics of Going Viral

Caltech researchers measure the rate of DNA transfer from viruses to bacteria

PASADENA, Calif.—Researchers at the California Institute of Technology (Caltech) have been able, for the first time, to watch viruses infecting individual bacteria by transferring their DNA, and to measure the rate at which that transfer occurs. Shedding light on the early stages of infection by this type of virus—a bacteriophage—the scientists have determined that it is the cells targeted for infection, rather than the amount of genetic material within the viruses themselves, that dictate how quickly the bacteriophage's DNA is transferred.

"The beauty of our experiment is we were able to watch individual viruses infecting individual bacteria,"says Rob Phillips, the Fred and Nancy Morris Professor of Biophysics and Biology at Caltech and the principal investigator on the new study. "Other studies of the rate of infection have involved bulk measurements. With our methods, you can actually watch as a virus shoots out its DNA."

The new methods and results are described in a paper titled "A Single-Molecule Hershey–Chase Experiment," which will appear in the July 24 issue of the journal Current Biology and currently appears online. The lead authors of that paper, David Van Valen and David Wu, completed the work while graduate students in Phillips's group.

In the well-known 1952 Hershey-Chase experiment, Alfred Hershey and Martha Chase of the Carnegie Institution of Washington in Cold Spring Harbor convincingly confirmed earlier claims that DNA—and not protein—was the genetic material in cells. To prove this, the researchers used bacteriophages, which are able to infect bacteria using heads of tightly bundled DNA coated in a protein shell. Hershey and Chase radiolabeled sulfur, contained in the protein shell but not in the DNA, and phosphorous, found in the DNA but not in the protein shell. Then they let the bacteriophages infect the bacterial cells. When they isolated the cells and analyzed their contents, they found that only the radioactive phosphorous had made its way into the bacteria, proving that DNA is indeed the genetic material. The results also showed that, unlike the viruses that infect humans, bacteriophages transmit only their genetic information into their bacterial targets, leaving their "bodies" behind.

"This led, right from the get-go, to people wondering about the mechanism—about how the DNA gets out of the virus and into the infected cell," Phillips says. Several hypotheses have focused on the fact that the DNA in the virus is under a tremendous amount of pressure. Indeed, previous work has shown that the genetic material is under more pressure within its protein shell than champagne experiences in a corked bottle. After all, as Phillips says, "There are 16 microns of DNA inside of a tiny 0.05 micron-sized shell. It's like taking 500 meters of cable from the Golden Gate Bridge and putting it in the back of a FedEx truck." 

Phillips's group wanted to find out whether that pressure plays a dominant role in transferring the DNA. Instead, he says, "What we discovered is that the thing that mattered most was not the pressure in the bacteriophage, but how much DNA was in the bacterial cell."

The researchers used a fluorescent dye to stain the DNA of two mutants of a bacteriophage known as lambda bacteriophage—one with a short genome and one with a longer genome—while that DNA was still inside the phage. Using a fluorescence microscope, they traced the glowing dye to see when and over what time period the viral DNA transferred from each phage into an E. coli bacterium. The mean ejection time was about five minutes, though that time varied considerably.

This was markedly different from what the group had seen previously when they ran a similar experiment in a test tube. In that earlier setup, they had essentially tricked the bacteriophages into ejecting their DNA into solution—a task that the phages completed in less than 10 seconds. In that case, once the phage with the longer genome had released enough DNA to make what remained inside the phage equal in length to the shorter genome, the two phages ejected DNA at the same rate. Therefore, Phillips's team reasoned, it was the amount of DNA in the phage that determined how quickly the DNA was transferred.

But Phillips says, "What was true in the test tube is not true in the cell." E. coli cells contain roughly 3 million proteins within a box that is roughly one micron on each side. Less than a hundredth of a micron separates each protein from its neighbors. "There's no room for anything else," Phillips says. "These cells are really crowded." 

And so, when the bacteriophages try to inject their DNA into the cells, the factor that limits the rate of transfer is how jam-packed those cells are. "In this case," Phillips says, "it had more to do with the recipient, and less to do with the pressure that had built up inside the phage."

Looking toward the future, Phillips is interested in using the methods he and his team have developed to study different types of bacteriophages. He also wants to investigate various molecules that could be helping to actively pull the viral DNA into the cells. In the case of a bacteriophage called T7, for instance, previous work has shown that the host cell actually grabs onto the DNA and begins making copies of its genes before the virus has even delivered all the DNA into the cell. "We're curious whether that kind of mechanism is in play with the lambda bacteriophage," Phillips says.

The current findings have implications for the larger question of how biomolecules like DNA and proteins cross membranes in general, and not just into bacteria. Cells are full of membranes that divide them into separate compartments and that separate entire cells from the rest of the world. Much of the business of cellular life involves getting molecules across those barriers. "This process that we've been studying is one of the most elementary examples of what you could call polymer translocation or getting macromolecules across membranes," Phillips says. "We are starting to figure out the physics behind that process."

In addition to Phillips, Van Valen, and Wu, the other authors on the Current Biology paper are graduate student Yi-Ju Chen; Hannah Tuson of the University of Wisconsin at Madison; and Paul Wiggins of the University of Washington. Van Valen is currently a medical student at UCLA's David Geffen School of Medicine, and Wu is an intern at the University of Chicago. The work was supported by funding from the National Science Foundation, a National Institutes of Health Medical Scientist Training Program fellowship, a Fannie and John Hertz Yaser Abu-Mostafa Graduate Fellowship, and an NIH Director's Pioneer Award.

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Kimm Fesenmaier
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Sarkis Mazmanian Discusses Benevolent Bacteria in Scientific American

There are trillions of bacteria living in our bodies, making up complex communities of microbes regulating processes like digestion and immunity. For Caltech biologist Sarkis Mazmanian, they also make up the focus of his research: understanding how the "good" bacteria promote human health. Featured in the cover story for the June issue of Scientific American, he makes a case for devoting more attention to the helpful bugs after years of scientific dedication to pathogens. "It goes against dogma to think that bacteria would make our immune systems function better," he says, in the article. "But the picture is getting very clear: the driving force behind the features of the immune system are commensals."

The magazine is available now on newsstands and the article, "The Ultimate Social Network," can be read online with a subscription.

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Decoding Worm Lingo

Caltech biologists discover that many nematode species make the same types of small-molecule pheromones

PASADENA, Calif.—All animals seem to have ways of exchanging information—monkeys vocalize complex messages, ants create scent trails to food, and fireflies light up their bellies to attract mates. Yet, despite the fact that nematodes, or roundworms, are among the most abundant animals on the planet, little is known about the way they network. Now, research led by California Institute of Technology (Caltech) biologists has shown that a wide range of nematodes communicate using a recently discovered class of chemical cues.

A paper outlining their studies—which were a collaborative effort with the laboratory of Frank C. Schroeder, assistant scientist at the Boyce Thompson Institute for Plant Research (BTI) of Cornell University—was published online April 12 in the journal Current Biology

Previous research by several members of this team had recently shown that a much-studied nematode, Caenorhabditis elegans, uses certain chemical signals to trade data. What was unknown was whether other worms of the same phylum "talk" to one another in similar ways.

But when the researchers looked at a variety of nematodes, they found the very same types of chemicals being combined and used for communication, says Paul Sternberg, the Thomas Hunt Morgan Professor of Biology at Caltech and senior author on the study. "It really does look like we've stumbled upon the letters or words of a universal nematode language, the syntax of which we don't yet fully understand," he says.

Nematodes are wide-ranging creatures; they have been found in hot springs, arctic ice, and deep-sea sediments. Many types of nematodes are harmless, or even beneficial, but others cause damage to plants and harm to humans and animals. Decoding the language of these worms could allow us to develop strategies to prevent the spread of unwanted nematode species, saving time and money for the agricultural and health-care industries.

"We can now say that many—maybe all—nematodes are communicating by secreting small molecules to build chemical structures called ascarosides," says Sternberg, whose past research in C. elegans found that those worms secrete ascarosides both as a sexual attractant and as a way to control the social behavior of aggregation. "It's really exciting and a big breakthrough that tells us what to look for and how we, too, might be able to communicate with this entire phylum of animals." 

Building upon Sternberg's previous findings, he and Andrea Choe, then a graduate student and now a postdoctoral scholar in biology at Caltech, decided to look for evidence of ascarosides in other species of nematodes. These included some parasitic organisms as well as some benign roundworm samples.

"I turned a section of Paul's lab into a parasite zoo, and people were both intrigued by it and terrified to come back there," says Choe. "One day they would see me cutting carrots to culture plant parasites, and the next I would be infecting mosquitoes or harvesting hookworms from rat intestines. We really tried to get as many different samples as we could."

Once they had cultured a sufficient number of different nematode species, the creatures were bathed in a liquid solution dubbed "worm water." This worm water collected the chemicals given off by the nematodes. The worms were then filtered out and sent to Schroeder's lab at BTI to be analyzed using a mass spectrometer—a tool used to deduce the chemical structure of molecules.

"When the results came back from BTI, showing that the same ascarosides were present in all the worm-water samples, I thought that they had made a mistake," says Choe. "It was a very surprising finding."

Using technology developed by Dima Kogan, a former graduate student at Caltech and coauthor of the paper, the researchers were also able to test the responses of various worms to particular ascarosides. Worms were placed on an agar plate, along with an experimental cue—a blend of ascarosides. Any action that might occur on the plates was then recorded; Kogan's software analyzed those recordings frame by frame, counting the number of worms that were either attracted or repelled by the given chemicals.

When asked about the development of the software, Choe explains that it all began when Kogan noticed that the current method involved counting worms by eye. "He was stunned that we would spend our time doing this," says Choe, "and he came up with this software in less than a week. It removed user bias, sped up our research 10-fold, and allowed us to study more chemicals and more species."

Next, the researchers will work to learn more about how the worms actually sense the ascarosides.

"Now that we know these chemicals are broadly present in nematodes, we want to find the genes that are responsible for the ability to respond to these chemicals," says Sternberg, who is also an investigator with the Howard Hughes Medical Institute. "That knowledge could open up a whole other angle, not just for dealing with the chemicals, but for actually interfering with those communication systems a little downstream by hitting the receivers."

The team also plans to continue deconstructing the language they have found among nematodes. For example, Sternberg wonders, how many different combinations of chemicals mean "food," or "mate," or "attack"? If the scientists can crack the code in terms of what different blends mean to different species, they can begin to interfere with the actions of the nematodes that wreak havoc across the world—leading to better eradication of plant pests, as well as human and animal parasites.

"There is only one known worm pheromone used in agriculture," says Choe. "It is time for us change that. This research could be a very big breakthrough on that front."

The Current Biology study, "Ascaroside Signaling is Widely Conserved Among Nematodes," was funded by a grant from the National Institutes of Health and was supported by the Howard Hughes Medical Institute. Additional authors on the study are Stephan H. von Reuss, from Schroeder's lab at BTI; Robin B. Gasser, from the University of Melbourne; and Edward G. Platzer, from UC Riverside.

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Determining a Stem Cell's Fate

Caltech biologists scour mouse genome for genes and markers that lead to T cells

PASADENA, Calif.—What happens to a stem cell at the molecular level that causes it to become one type of cell rather than another? At what point is it committed to that cell fate, and how does it become committed? The answers to these questions have been largely unknown. But now, in studies that mark a major step forward in our understanding of stem cells' fates, a team of researchers from the California Institute of Technology (Caltech) has traced the stepwise developmental process that ensures certain stem cells will become T cells—cells of the immune system that help destroy invading pathogens.

"This is the first time that a natural developmental process has been dissected in such detail, going from step to step to step, looking at activities of all the genes in the genome," says Ellen Rothenberg, the principal investigator on the study and Albert Billings Ruddock Professor of Biology at Caltech. "It means that in genetic terms, there is virtually nothing left hidden in this system."

The study was led by Jingli A. Zhang, a graduate student in Rothenberg's lab, who is now a postdoctoral scholar at Caltech. The group's findings appear in the April 13 issue of the journal Cell.

The researchers studied multipotent hematopoietic precursor cells—stem-cell-like cells that express a wide variety of genes and have the capability to differentiate into a number of different blood-cell types, including those of the immune system. Taking into consideration the entire mouse genome, the researchers pinpointed all the genes that play a role in transforming such precursor cells into committed T cells and identified when in the developmental process they each turn on.  At the same time, the researchers tracked genes that could guide the precursor cells to various alternative pathways.  The results showed not only when but also how the T-cell-development process turned off the genes promoting alternative fates.

"We were able to ask, 'Do T-cell genes turn on before the genes that promote some specific alternative to T cells turn off, or does it go in the other order? Which genes turn on first? Which genes turn off first?'" Rothenberg explains. "In most genome-wide studies, you rarely have the ability to see what comes first, second, third, and so on, in a developmental progression. And establishing those before-after relationships is absolutely critical if you want to understand such a complicated process."

The researchers studied five stages in the cascade of molecular events that yields a T cell—two before commitment, a commitment stage, and then two following commitment. They identified the genes that are expressed throughout those stages, including many that code for regulatory proteins, called transcription factors, which turn particular genes on or off. They found that a major regulatory shift occurs between the second and third stages, when T-cell commitment sets in. At that point, a large number of the transcription factors that activate genes associated with uncommitted stem cells turn off, while others that activate genes needed for future steps in T-cell development turn on.

The researchers looked not only at which genes are expressed during the various stages but also at what makes it possible for those genes to be expressed at that particular time. One critical component of regulation is the expression of transcription-factor genes themselves. Beyond that, the researchers were interested in identifying control sequences—the parts of genes that serve as docking sites for transcription factors. These sequences are often very difficult to identify in mice and humans using classical molecular-biology techniques; scientists have spent as many as 10 years trying to create a comprehensive map of the control sequences for a single gene. 

To create a map of likely control sequences, Zhang studied epigenetic markers. These are chemical modifications, such as those that change the way the DNA is bundled. They become associated with particular regions of DNA as a result of the action of transcription factors and can thereby affect how easy or hard it is for a neighboring gene to be turned on or off. By identifying DNA regions where epigenetic markers are added or removed, Rothenberg's group has paved the way for researchers to identify control sequences for many of the genes that turn on or off during T-cell development.

In some ways, Rothenberg says, her team is taking a backward approach to the problem of locating these control sequences. "What we're saying is, if we can tell that a gene is turned on at a certain point in terms of producing RNA, then we should also be able to look at the DNA sequences right around it and ask, 'Is there any stretch of DNA sequence that adds or loses epigenetic markers at the same time?'" Rothenberg says. "If we find it, that can be a really hot candidate for the control sequences that were used to turn that gene on." 

Two methodologies have made it possible to complete this work. First, ultra-high-throughput DNA sequencing was used to identify when major changes in gene expression occur along the developmental pathway. This technique amplifies DNA sequences taken throughout millions of cell samples, puts all of the bits in order, compares them to the known genome sequence (for mice, in this case), and identifies which of the various genes are enriched, or found in greater numbers. Those that are enriched are the ones most likely to be expressed. The team also used a modified version of this sequencing technique to identify the parts of the genome that are associated with particular epigenetic markers. Coauthor Barbara Wold, Caltech's Bren Professor of Molecular Biology, is an expert in these so-called "next-generation" deep-sequencing technologies and provided critical inspiration for the study.

A second important methodology involved an in vitro tissue-culture system developed in the lab of Juan Carlos Zúñiga-Pflücker of the University of Toronto, which enabled the Caltech researchers to mass-produce synchronized early T-cell precursors and to see the effect of altered conditions on individual cells in terms of producing T cells or other cells.

Zhang is lead author of the paper in Cell, titled "Dynamic transformations of genome-wide epigenetic marking and transcriptional control establish T cell identity." In addition to Zhang, Rothenberg, and Wold, Brian Williams, a senior scientific researcher at Caltech, was also a coauthor. Another coauthor, developmental biologist Ali Mortazavi, was part of Wold's lab and was also associated with the lab of Paul Sternberg when the work was completed; he is now an assistant professor at the University of California, Irvine.

The work was supported by the Beckman Institute, the Millard and Muriel Jacobs Genetics and Genomics Laboratory, the L.A. Garfinkle Memorial Laboratory Fund, the Al Sherman Foundation, the Bren Professorship, the A.B. Ruddock Professorship, and grants from the National Institutes of Health.

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Kimm Fesenmaier
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Alexander Varshavsky Awarded Otto Warburg Medal

Alexander Varshavsky, Caltech's Howard and Gwen Laurie Smits Professor of Cell Biology, has been awarded the Otto Warburg Medal of the German Society for Biochemistry and Molecular Biology (GBM). The medal is considered to be the highest German award for biochemists and molecular biologists.

Varshavsky's main recognized contribution was the fundamental discovery, in the 1980s, of biological regulation by intracellular protein degradation and its central importance in cellular physiology. This discovery by the Varshavsky laboratory involved the understanding, through genetic and biochemical insights, of the biological functions of the ubiquitin system, a major proteolytic circuit in living cells. Ubiquitin is a small protein that is present in cells either as a free protein or as a part of complexes with many other proteins. The association of ubiquitin with cellular proteins marks them for degradation or other metabolic fates. Through its ability to destroy specific proteins, the ubiquitin system plays a major role in cell growth and differentiation, DNA repair, regulation of gene expression, and many other biological processes.

"Alexander Varshavsky's research on ubiquitin-dependent intracellular protein degradation has revolutionised that area of research," Professor Irmgard Sinning, president of GBM, said in the citation. "We owe to him a series of discoveries with a tremendous impact on cell biology and helping us to better understand and study numerous diseases."

The recipient of numerous awards, including most recently the 2012 King Faisal International Prize for Science, Varshavsky earned his BS from Moscow State University in 1970 and his PhD from the Institute of Molecular Biology in 1973. He has been Smits Professor at Caltech since 1992.

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Allison Benter
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Do You Hear What I Hear?

Caltech biologists locate brain's processing point for acoustic signals essential to human communication

PASADENA, Calif.—In both animals and humans, vocal signals used for communication contain a wide array of different sounds that are determined by the vibrational frequencies of vocal cords. For example, the pitch of someone's voice, and how it changes as they are speaking, depends on a complex series of varying frequencies. Knowing how the brain sorts out these different frequencies—which are called frequency-modulated (FM) sweeps—is believed to be essential to understanding many hearing-related behaviors, like speech. Now, a pair of biologists at the California Institute of Technology (Caltech) has identified how and where the brain processes this type of sound signal.

Their findings are outlined in a paper published in the March 8 issue of the journal Neuron.

Knowing the direction of an FM sweep—if it is rising or falling, for example—and decoding its meaning, is important in every language. The significance of the direction of an FM sweep is most evident in tone languages such as Mandarin Chinese, in which rising or dipping frequencies within a single syllable can change the meaning of a word.

In their paper, the researchers pinpointed the brain region in rats where the task of sorting FM sweeps begins.

"This type of processing is very important for understanding language and speech in humans," says Guangying Wu, principal investigator of the study and a Broad Senior Research Fellow in Brain Circuitry at Caltech. "There are some people who have deficits in processing this kind of changing frequency; they experience difficulty in reading and learning language, and in perceiving the emotional states of speakers. Our research might help us understand these types of disorders, and may give some clues for future therapeutic designs or designs for prostheses like hearing implants."

The researchers—including co-author Richard I. Kuo, a research technician in Wu's laboratory at the time of the study (now a graduate student at the University of Edinburg)—found that the processing of FM sweeps begins in the midbrain, an area located below the cerebral cortex near the center of the brain—which, Wu says, was actually a surprise.

"Some people thought this type of sorting happened in a different region, for example in the auditory nerve or in the brain stem," says Wu. "Others argued that it might happen in the cortex or thalamus. "

To acquire high-quality in-vivo measurements in the midbrain, which is located deep within the brain, the team designed a novel technique using two paired—or co-axial—electrodes. Previously, it had been very difficult for scientists to acquire recordings in hard-to-access brain regions such as the midbrain, thalamus, and brain stem, says Wu, who believes the new method will be applicable to a wide range of deep-brain research studies.

In addition to finding the site where FM sweep selectivity begins, the researchers discovered how auditory neurons in the midbrain respond to these frequency changes. Combining physical measurements with computational models confirmed that the recorded neurons were able to selectively respond to FM sweeps based on their directions. For example, some neurons were more sensitive to upward sweeps, while others responded more to downward sweeps.

"Our findings suggest that neural networks in the midbrain can convert from non-selective neurons that process all sounds to direction-selective neurons that help us give meanings to words based on how they are spoken. That's a very fundamental process," says Wu.  

Wu says he plans to continue this line of research, with an eye—or ear—toward helping people with hearing-related disorders. "We might be able to target this area of the midbrain for treatment in the near future," he says.

The Neuron study, "The Generation of Direction Selectivity in the Auditory System," was funded by grants from the Broad Fellows Program in Brain Circuitry of the Broad Foundation and Caltech.

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Katie Neith
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