Rothenberg Wins Feynman Prize

The 2016 Richard P. Feynman Prize for Excellence in Teaching has been awarded to Ellen Rothenberg, the Albert Billings Ruddock Professor of Biology.

Established in 1993, the Feynman Prize annually honors "a professor who demonstrates, in the broadest sense, unusual ability, creativity, and innovation in undergraduate and graduate classroom or laboratory teaching." Rothenberg, who has been at Caltech since joining the faculty as an assistant professor in 1982, was nominated for the prize by her students, who cited qualities such as her passion for teaching and her engagement with students as the reason for their nominations.

Rothenberg investigates the regulatory mechanisms that control blood stem cell differentiation and the development of T lymphocytes—white blood cells that play an important role in immunity. Not surprisingly, when she began at Caltech, her first teaching assignment was Immunology (Bi 114), a course that she continued to teach for 25 years, consistently receiving high ratings from her students in her teaching-quality feedback reports. In 1989, Rothenberg also introduced Caltech's first course on the molecular biology of blood development, Hematopoiesis: A Developmental System (Bi 214)—a course that she still teaches every other year.

Rothenberg recently was instrumental to changes made to the introductory biology courses at Caltech. "I was the chair of the Curriculum Committee, and I noticed that there were issues that arose for both students and faculty with the first two introductory courses," she says. Beginning in 2008, she began redeveloping and teaching these introductory courses, Cell Biology (Bi 9) and then molecular biology (Bi 8). A student's first two terms at Caltech are mandatory pass/fail, "and we discovered that the students are actually really excited to do something hard when it's on a pass/fail basis," she explains.

In a letter of nomination, one of Rothenberg's students said that she appreciated the challenge to learn more complicated material in an introductory course. "In her course, Professor Rothenberg emphasizes important concepts about molecular biology; however, she also takes time to explore higher-level concepts with incredible enthusiasm," the student said. "This introduced me to the many complex systems I could learn about while showing me how exciting biological research is. I also sit on the Curriculum Committee, which she leads, and I have seen how she constantly returns to the idea of what will help students learn best and what will train them effectively."

Another student who nominated Rothenberg wrote that "… she showed students that, contrary to what they might have heard, biology was not simply a 'memorization game,' but rather a logic puzzle. By slowly introducing us to different research techniques, she allowed us to see how we could pose and answer questions in biology ourselves."

In addition to challenging her students to learn in a new way, Rothenberg says that these introductory courses also challenged her to teach differently. Because introductory courses have larger class sizes, she says it was inherently more difficult to get to know her students. So, she found ways to connect with her students outside of class time. "She spends a lot of time with her students," one student said in a nomination, "even actively participating in recitation sections with her TAs, an unusual task for professors. She strives to improve her class every year."

Previously, Rothenberg was awarded the Biology Undergraduate Students Advisory Council Award for excellence in teaching four times, the Ferguson Prize for Undergraduate Teaching twice, and the ASCIT Award for Undergraduate Teaching twice. In addition, she has chaired the divisional Curriculum Committee for the past several years, working to rationalize the biology curriculum and to put the best teachers in place for each course. As part of her work on the Curriculum Committee, she interacts closely with the Biology Undergraduate Students Advisory Council.

"Winning this award and being recognized at an institutional level…it means a lot to me. And I'm also really humbled that I'm the first biologist ever to get the Feynman Prize," she says. "I love teaching. The greatest gift you can give someone is to share your understanding with them and to help them develop their own understanding. That incredible connection between the way you appreciate the complexity of the world and the way you can give students the tools to see things that you never saw before—it's really beautiful. And the fact that this institute has a way of valuing that is really wonderful," she adds.

The Feynman Prize has been endowed through the generosity of Caltech Associates Ione and Robert E. Paradise and an anonymous local couple. Some of the most recent winners of the Feynman Prize include Kevin Gilmartin, professor of English; Steven Frautschi, professor of theoretical physics, emeritus; and Paul Asimow, professor of geology and geochemistry.

Nominations for next year's Feynman Prize for Excellence in Teaching will be solicited in the fall. Further information about the prize can be found on the Provost's Office website.

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Rothenberg Wins Feynman Prize
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The 2016 Richard Feynman Prize for Excellence in Teaching has been awarded to Ellen Rothenberg, the Albert Billings Ruddock Professor of Biology.

Learning to Program Cellular Memory

What if we could program living cells to do what we would like them to do in the body? Having such control—a major goal of synthetic biology—could allow for the development of cell-based therapies that might one day replace traditional drugs for diseases such as cancer. In order to reach this long-term goal, however, scientists must first learn to program many of the key things that cells do, such as communicate with one another, change their fate to become a particular cell type, and remember the chemical signals they have encountered.

Now a team of researchers led by Caltech biologists Michael Elowitz, Lacramioara Bintu, and John Yong (PhD '15) have taken an important step toward being able to program that kind of cellular memory using tools that cells have evolved naturally. By combining synthetic biology approaches with time-lapse movies that track the behaviors of individual cells, they determined how four members of a class of proteins known as chromatin regulators establish and control a cell's ability to maintain a particular state of gene expression—to remember it—even once the signal that established that state is gone.

The researchers reported their findings in the February 12 issue of the journal Science.

"We took some of the most important chromatin regulators for a test-drive to understand not just how they are used naturally, but also what special capabilities each one provides," says Elowitz, a professor of biology and bioengineering at Caltech and an investigator with the Howard Hughes Medical Institute (HHMI). "We're playing with them to find out what we can get them to do for us."

Rather than relying on a single protein to program all "memories" of gene expression, animal cells use hundreds of different chromatin regulators. These proteins each do basically the same thing—they modify a region of DNA to alter gene expression. That raises the question, why does the cell need all of these different chromatin regulators? Either there is a lot of redundancy built into the system or each regulator actually does something unique. And if the latter is the case, synthetic biologists would like to know how best to use these regulators as tools—how to select the ideal protein to achieve a certain effect or a specific type of cellular memory.

Looking for answers, the researchers turned to an approach that Elowitz calls "build to understand." Rather than starting with a complex process and trying to pick apart its component pieces, the researchers build the targeted biological system in cells from the bottom up, giving themselves a chance to actually watch what happens with each change they introduce.

In this case, that meant sticking different chromatin regulators—four gene-silencing proteins—down onto a specific section of DNA and seeing how each behaved. In order to do that the researchers engineered cells so that adding a small molecule would cause one of the gene-silencing regulators to bind to DNA near a particular gene that codes for a fluorescent protein. By tracking fluorescence in individual cells, the researchers could readily determine whether the regulator had turned off the gene. The researchers could also release the regulator from the DNA and see how long the gene remembered its effect.

Although there are hundreds of chromatin regulators, they can be categorized into about a dozen broader classes. For this study, the researchers tested regulators from four biochemically diverse classes.

"We tried a variety to see if different ones give you different types of behavior," explains Bintu. "It turns out they do."

For a month at a time, the researchers used microscopy or flow cytometry to observe the living cells, using cell-tracking software that they wrote and time-lapse movies to watch individual cells grow and divide. In some cases, after a regulator was released, the cells and their daughter cells remained dark for days and then lit back up, indicating that they remembered the modification transiently. In other cases, the cells never lit back up, indicating more permanent memory.

After modification, the genes were always in one of three states—"awake" and actively making protein, "asleep" and inactive but able to wake up in a matter of days, or "in a coma" and unable to be awakened within 30 days. Within an individual cell, the genes were always either completely on or off.

That led the researchers to the surprising finding that the regulators control not the level or degree of expression of a particular gene in an individual cell, but rather how many cells in a population have that gene on or off.

"You're controlling the probability that something is on or off," says Elowitz. "We think that this is something that's very useful generally in a multicellular organism—that in many cases, the organism may want to tell cells, 'I just want 30 percent of you to differentiate. You don't all need to do it.' This chromatin regulation system seems ready-made for orders like those."

In addition, the researchers found that the type of memory imparted by each of the four chromatin regulators was different. One produced permanent memory, turning off the gene and putting a fraction of cells into a coma for the full 30 days. One yielded short-term memory, with the cells immediately waking up. "The really interesting thing we found is that some of the regulators give this type of hybrid memory where some of the cells awaken while a fraction of the cells remain in a deep coma," says Bintu. "How many are in the coma depends on how long you gave the signal—how long the chromatin regulator stayed attached."

Going forward, the group plans to study additional chromatin regulators in the same manner, developing a better sense of the many ways they are used in the cell and also how they might work in combination. In the longer term they want to put these proteins together with other cellular components and begin programming more complex developmental behavior in synthetic circuits.

"This is a step toward realizing this emerging vision of programmable cell-based therapies," says Elowitz. "But we are also answering more basic research questions. We see these as two sides of the same coin. We're not going to be able to program cells effectively until we understand what capabilities their core pathways provide. "

Additional Caltech authors on the paper, "Dynamics of epigenetic regulation at the single-cell level," include Yaron E. Antebi and Kayla McCue (BS '15). Yasuhiro Kazuki, Narumi Uno, and Mitsuo Oshimura of Tottori University in Japan are also coauthors. The work was supported by the Defense Advanced Research Projects Agency, the Human Frontier Science Program, the Jane Coffin Childs Memorial Fund for Medical Research, the Beckman Institute at Caltech, the Burroughs Wellcome Fund, and HHMI.

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Kimm Fesenmaier
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Learning to Program Cellular Memory
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Combining synthetic biology approaches with time-lapse movies, biologists have determined how some proteins shape a cell's ability to remember signals.

New CCE Leadership Chair Honors the Past and Supports the Future

A new leadership chair in Caltech's Division of Chemistry and Chemical Engineering (CCE) will amplify the Institute's support of its scholars' freedom to pursue the most interesting and challenging lines of inquiry. Established through a $10 million gift from a couple who wish to remain anonymous, the chair will be named in honor of the late Norman Davidson, a longtime Caltech faculty member whose scientific contributions represented in part the beginnings of molecular biology. 

"Through this gift, CCE can maintain its excellence and move forward in new frontiers in chemistry," says Jacqueline K. Barton, Caltech's Arthur and Marian Hanisch Memorial Professor of Chemistry and inaugural holder of the Norman Davidson Leadership Chair.

Read the full story at giving.caltech.edu

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A new leadership chair in the Division of Chemistry and Chemical Engineering (CCE) will amplify the Institute’s support of its scholars’ freedom.

Caltech Bioethics Forum: HeLa Cells in the Lab

Henrietta Lacks died of cervical cancer in 1951 and, ever since, samples of her uniquely immortal cancerous cells have been used in scientific research, sparking great leaps in medical knowledge.

But the cells—taken without her knowledge or consent—have also fueled controversy and called into question the ethical underpinnings of the research they made possible. Her story, made famous in the book, The Immortal Life of Henrietta Lacks, by science writer Rebecca Skloot, underscores the continuing need—65 years after her death—for society to find a way to balance the advancement of medical knowledge with the protection of individual rights.

At a February 22 bioethics forum at Baxter Lecture Hall, Caltech president Thomas F. Rosenbaum introduced a panel of Caltech faculty that examined the ethics of using Lacks's cells—known as HeLa cells—along with issues of privacy, informed consent, and who profits from the technologies her cells engendered. Caltech trustee Ronald L. Olson moderated the panel of Caltech faculty, which featured David Baltimore, President Emeritus and the Robert Andrews Millikan Professor of Biology; Ellen Rothenberg, the Albert Billings Ruddock Professor of Biology; Barbara J. Wold, the Bren Professor of Molecular Biology; and Changhuei Yang, professor of electrical engineering, bioengineering, and medical engineering.

The evening event revisited a topic that incoming freshmen had tackled earlier in the academic year in roundtable discussions of the book, which the students had been asked to read prior to their arrival at Caltech.

"HeLa cells were a miracle," said Baltimore, who has used them in his research since 1962. After noting their incalculable value—and how rare it was to have found cells that underwent the specific mutations that conferred their ability to divide indefinitely in culture—he brought the discussion back to the question of who owns the cells.

"So it seems to me the question is, what rights does she have as a consequence of this rare, basically random event, that made her cells different than anybody else's cells? . . . We are the product of a genetic lottery. What rights do we get as a consequence of our particular genes? Everyone else around us has a representation of those same genes, but not identical. What is ownership in this case?"

Rothenberg discussed how the cells have enabled key advances in molecular biology, stem cell research, and immunology—advances that would have been considered "complete science fiction fantasy" when Lacks was alive. Because the innovations her cells made possible would have been impossible to foresee, questions naturally arise as to whether Lacks could have understood the ramifications of her consent, had it been sought.

The structure of DNA was only discovered two years after Lacks' death, and the revolution in molecular biology that followed completely transformed the possibilities for use of any human cells that were able to grow in culture. "How could she possibly have given informed consent? In the case of a rapidly advancing field like molecular biology, there's no way she could have been asked at that time what she was really consenting to," Rothenberg said.

When the conversation returned to ownership of a patient's cells, and who should profit from their use, Rothenberg pointed out that simply saying the patient owns them, period, could generate a raft of unintended consequences. For example, medical institutions might, for legal liability reasons, refuse to accept certain tissue samples, hampering the delivery of personalized medicine to patients. Equally disturbing, she says, would be the possibility that the commercially valuable products of cells might incentivize individuals to view a cancer-patient relative "as a possible cash cow, and sell their tissues in the hopes of winning the lottery. . . . You definitely don't want people to be in a position where they or their family members want to sell parts of their body because they're starving."

Wold said her research seldom involves HeLa cells but, she added, "that doesn't mean I'm not an avid consumer of what's been learned over 60 years of studying them." She hailed the "beautiful science" the cells have engendered, but lamented the scientific community's repeated failings in communicating with and involving the Lacks family over the years as to how the cells were being used and what was being learned from them. For example, she said, teams of researchers in Germany and at the University of Washington sequenced and published the HeLa genome in 2013 and made the information freely available worldwide. In doing so, however, they made portions of the family's genome public—without thinking to seek the family's approval or tell the family what was happening. The research community "quickly recognized this as a catastrophe," said Wold, prompting the creation of a board that includes Lacks family members and now regulates access to the data.

Such ethical considerations continued during the event's Q&A, which stirred discussion about such critical questions as how to address medical privacy when one family member's consent might make public another family member's information, and whether proposed consent rules might jeopardize access to older cell lines that were obtained prior to a stricter consent regime.

Yang, whose lab has used HeLa cells since 2008, said he only recently learned about the ethical concerns around their provenance, adding, "Honestly I was quite surprised to find there were all these [controversies]. . . . As an outsider to American culture—I actually grew up in Singapore—my instinct would be that the DNA is a common good, not personal property. If my cells would be useful for research, I would gladly give them up without any expectations."

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Caltech Bioethics Forum: HeLa Cells in the Lab
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Caltech faculty examine the ethics of using Henrietta Lacks’s cells—covering issues of privacy, informed consent, and who profits from the technologies her cells engendered.
Friday, April 15, 2016
Beckman Institute Auditorium – Beckman Institute

A Celebration of Eric Davidson: Visionary Insights into the Genomic Control of Development and Evolution

Gradinaru and Benardini Receive Presidential Early Career Awards

Viviana Gradinaru (BS '05), an assistant professor of biology and biological engineering, and James Benardini, a planetary protection engineer at JPL, have been named as recipients of the 2016 Presidential Early Career Award for Scientists and Engineers (PECASE).

The PECASE awards were created to foster innovative developments in science and technology, increase awareness of careers in science and engineering, give recognition to the scientific missions of participating agencies, enhance connections between fundamental research and many of the grand challenges facing the nation, and highlight the importance of science and technology for America's future. The award is the highest honor bestowed by the United States government on science and engineering professionals in the early stages of their independent research careers.

Viviana Gradinaru is an assistant professor of biology and biological engineering as well as the faculty director of the Beckman Institute Center for CLARITY, Optogenetics, and Vector Engineering Research (CLOVER). Her work focuses on developing technologies such as optogenetics (using light to control genetically modified cells) and tissue clearing (that can render rodent tissues and bodies transparent via PARS CLARITY). Most recently, she and her team have discovered how to modify the protein shell of a harmless virus to successfully enter the adult mouse brain from the bloodstream—crossing the so-called blood-brain barrier—and deliver genes to cells of the nervous system. Gradinaru employs these technologies for mapping and modulating brain networks to understand and develop therapies for neurological diseases.

James (Nick) Benardini is the planetary protection lead on the InSight and Mars 2020 missions. He and his colleagues study how to minimize microbial and other biological contamination on outgoing space missions. This involves the use of clean rooms and microbial reduction modalities in addition to looking for genetic traces on samples collected from spacecraft and spacecraft-associated surfaces.

In addition to Gradinaru, three other Caltech alumni were named as 2016 PECASE recipients: Alon Gorodetsky (PhD '09), Jon Simon (BS '04), and Tammy Ma (BS '05).

The winners will receive their awards at a ceremony in Washington, D.C., this spring.

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Viviana Gradinaru and James Benardini have been named as recipients of the 2016 Presidential Early Career Award for Scientists and Engineers.
Thursday, March 17, 2016
Beckman Institute Auditorium – Beckman Institute

New Horizons in Biology 2016 Faculty Hiring Symposium

Studying Memory's 'Ripples'

Caltech neuroscientists have looked inside brain cells as they undergo the intense bursts of neural activity known as "ripples" that are thought to underlie memory formation. 

During ripples, a small fraction of brain cells, or neurons, fire synchronously in area CA1, a part of the hippocampus that is thought to be an important relay station for memories. "During a ripple, about 10 percent of the neurons in CA1 are activated, and these active neurons all fire within a tenth of a second," says Caltech graduate student Brad Hulse. "Two big questions have been: How do the remaining 90 percent of CA1 neurons stay quiet? And what is synchronizing the firing of the active neurons?"

In a new study, published online on February 17 in the journal Neuron, Hulse and his colleagues used a novel approach to show how the combination of excitatory and inhibitory inputs to CA1 work together to synchronize the firing of active neurons while keeping most neurons silent during ripples.

"For a long time, people studied these events by placing an electrode outside of a cluster of neurons. These extracellular recordings can tell you about the output of a group of brain cells, but they tell you very little about the inputs they're receiving," says study coauthor and Caltech research scientist Evgueniy Lubenov.

The Caltech scientists combined extracellular recording with a technique to look inside a neuron during ripples. They used fine glass pipettes with tips thinner than a tenth of the width of a human hair to measure directly the voltage difference, or "electrical potential," across the cellular membrane of individual neurons in awake mice.

Employing these two techniques in tandem allowed the scientists to monitor the activity inside a single neuron while still observing how the larger network was behaving. This in turn enabled them to piece together how excitatory inputs from CA3, a hippocampal region where memories are formed, affect the output of brain cells called pyramidal neurons in CA1. These neurons are important for transferring newly coded memories to other brain areas such as the neocortex for safekeeping and long-term storage. Ripples are thought to be the mechanism by which this transfer occurs.

The team discovered that the membrane potential of CA1 pyramidal cells increases during ripples. Surprisingly, this increase is relatively constant and independent of the strength of the input from CA3. For this to be the case, the direct excitation from CA3 must be balanced by proportional inhibition. The source of this inhibition is presumed to be a class of brain cells called feedforward interneurons, which receive direct inputs from CA3 and inhibit CA1 pyramidal cells.

"There seems to be a circuit mechanism that balances excitation and inhibition, so that for most neurons, these two forces cancel out," says study leader Thanos Siapas, professor of computation and neural systems at Caltech.

Without a balanced inhibition, all of the neurons in CA1 could fire when the excitatory input is large enough. "This could cause runaway excitation and possibly trigger a seizure," says Hulse, who is the first author of the new study.

The team's finding explains why most CA1 pyramidal neurons remain silent during ripples, but it raises two important questions: Why do any neurons fire at all? And what controls the precise timing of those that do fire?

The Caltech researchers found that active neurons receive a much stronger excitatory input from CA3 than silent neurons do—one that is large enough to overcome the balancing inhibition. This large excitation originates from CA3 neurons with particularly strong connections to the active CA1 neurons. These connections are believed to be modified during behavior to encode memories.

Hence, it is the specific identity of CA3 neurons, rather than their sheer number, that is responsible for making CA1 neurons fire, the researchers say. This system might seem overly complex and redundant, but the end result is a flexible circuit—an ever-changing mosaic of active and inactive pyramidal neurons. "It's a shifting mosaic, but it's one that is dependent on the organism's memories and experience," Siapas says.

How do ripples exert their influence on the rest of the brain? The membrane potential of each neuron oscillates very rapidly during ripples to synchronize the firing of cells to within a few thousandths of a second. "By coordinating their activities, the CA1 neurons are maximizing the impact of their output on downstream areas of the brain. The overall effect is more powerful than if each neuron fired independently," Lubenov says. "It is the difference between clapping independently or in unison with others at a concert. The effect in the latter case is stronger, even with the same number of people applauding."

Neuroscientists previously thought that these fast oscillations were generated by rhythmic firing of inhibitory neurons, but the Caltech team showed that this cannot be the whole story. "Our experiments suggest that it is the interplay between rhythmic excitation and inhibition that drives these fast oscillations," Hulse says.

The paper, "Membrane Potential Dynamics of CA1 Pyramidal Neurons during Hippocampal Ripples in Awake Mice," is also coauthored by Laurent C. Moreaux, a research scientist at Caltech. Funding for the work was provided by the Mathers Foundation, the Gordon and Betty Moore Foundation, the National Institutes of Health, and the National Science Foundation. 

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Studying Memory's 'Ripples'
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Neuroscientists look inside brain cells undergoing the bursts of activity, or "ripples," that underlie memory formation

Caltech Biologists Identify Gene That Helps Regulate Sleep

Caltech biologists have performed the first large-scale screening in a vertebrate animal for genes that regulate sleep, and have identified a gene that when overactivated causes severe insomnia. Expression of the gene, neuromedin U (Nmu), also seems to serve as nature's stimulant—fish lacking the gene take longer to wake up in the morning and are less active during the day.

The findings improve our understanding of how sleep is regulated—a process that we know surprisingly little about despite its clear importance. In the long term, the results suggest Nmu as a potential candidate for new therapies to address sleep disorders.

A paper describing the new screening process and its results appears in the February 17, 2016, issue of the journal Neuron. David Prober, assistant professor of biology at Caltech, began the work as a postdoctoral fellow at Harvard University, and has continued the work at Caltech since 2009. The lead authors on the paper are Cindy Chiu (PhD '14), a former graduate student in Prober's lab, and Jason Rihel, who collaborated with Prober at Harvard and now has his own lab at University College London.

"Sleep is a mysterious process," says Prober. "We spend a third of our lives doing it, and every animal with a complex nervous system seems to do it, so it must be important. But we still don't understand why we do it or how it's regulated."

Genetic screens are a powerful method that can help identify the genetic basis of such behaviors. They typically involve mutating the DNA of thousands of animals, raising them, identifying any resulting physical or behavioral differences, and determining which altered gene produced each mutation. This approach works well for simple model organisms, such as fruit flies and worms, because their anatomy is relatively simple and it is easy to raise large numbers of them, but is far more difficult in vertebrates, such as rodents.

Recently, zebrafish have emerged as a valuable vertebrate model system for studying sleep. Compared to a mouse, the small, striped fish are much easier to work with. Many can be raised in a small space (a larva is about 4 millimeters long, about the same size as a fruit fly); they develop quickly, exhibiting complex behaviors, such as hunting, by the time they are five days old; and they are transparent during their embryonic and larval stages, making it simpler for researchers to track what is happening inside their brains. Like humans, zebrafish sleep for consolidated periods of time at night. Furthermore, Prober says, "anatomical and molecular similarities between zebrafish and mammalian brains suggest that the basic neural circuits regulating sleep in zebrafish are likely conserved in mammals."

Rather than mutating the DNA and testing which functions were lost, the researchers used a gain-of-function approach in the new study. Just after fertilization, when the zebrafish embryos were still single cells, the researchers injected them with a DNA molecule, called a plasmid, carrying a gene that was inserted into the genome of some of the cells in each fish. In particular, they wanted to test genes that are predicted to encode for secreted proteins—those, like neuropeptides, that cells make and then release. Many of the genes that have been identified as being involved in sleep encode neuropeptides.

Using a genetic switch called a heat-shock promoter, which turns on only when the fish are heated to about 37 degrees Celsius, the biologists were able to control when the fish expressed each inserted gene. They kept the switch on long enough for the fish to overexpress each gene, making many copies of the products. Then they used computerized video trackers to monitor the fish for several days to see which genes affected sleep.

Next, the researchers made transgenic zebrafish for each of the genes that had demonstrated strong effects on sleep in the genetic screen. That labor-intensive approach gave them zebrafish in which all cells overexpressed a particular gene in response to heat shock, providing more robust results.

In the end, the most significant change resulted from overexpression of Nmu, a gene that is also found in mammals and is expressed in a part of the brain called the hypothalamus.

"After heat shock, the fish that overexpress Nmu are much more active both during the day and at night," says Prober. "The fish almost don't sleep at all the night following the heat shock—so they display a very profound form of insomnia."

When the researchers mutated the zebrafish so that they did not have Nmu, the larvae were less active during the day. Adult fish without the gene were particularly sluggish first thing in the morning.

Like humans, zebrafish normally start to wake up at the end of the night and then become much more active when the lights turn on. "The fish without Nmu are defective in this anticipation of dawn," says Prober. "So it seems that this gene is particularly important for the transition from nighttime sleep to daytime wakefulness."

To explore how Nmu promotes wakefulness, the researchers first investigated the gene's role in a stress response pathway known as the hypothalamic-pituitary-adrenal (HPA) axis. Researchers had previously shown Nmu to be involved in arousal caused by stressful situations and hypothesized that it was involved in activating the HPA axis. However, Prober and his colleagues found that Nmu suppressed sleep to the same extent in zebrafish mutants lacking a protein called the glucocorticoid receptor, which is necessary for HPA axis signaling, as it did in fish with a functional glucocorticoid receptor, suggesting that the gene does not act through the HPA axis.

The researchers then went back to the drawing board and asked which neurons in the brain became activated as a result of Nmu overexpression. Using a technique that labels activated neurons, they saw strong activation of a handful of cells that express a gene called corticotrophin-releasing hormone (CRH) in the brainstem.

"That was surprising because CRH is the gene that initiates the HPA axis response, but the cells that do that are in the hypothalamus, a different part of the brain, and they aren't activated when we overexpress Nmu," says Prober. "It's another population of CRH cells in the brainstem that are activated by Nmu overexpression."

A low dose of a drug that blocks CRH signaling completely blocked the wake-promoting effect of Nmu overexpression in zebrafish, the researchers found, whereas a higher dose also reduced wakefulness in normal fish.

"So not only is CRH signaling required for the effects of Nmu on behavior, it's also required for normal levels of activity," explains Prober.

Several wake-promoting or sleep-promoting genes and neurons have been identified, he notes. However, scientists still do not know which are the relevant ones for causing sleep disorders in humans. "Our study suggests that Nmu could be a good gene to look into."

Additional Caltech authors on the paper, "A Zebrafish Genetic Screen Identifies Neuromedin U as a Regulator of Sleep/Wake States," are Daniel A. Lee, Chanpreet Singh, Eric A. Mosser, Shijia Chen, Viveca Sapin, Uyen Pham, Jae Engle, Brett J. Niles, Christin J. Montz, and Sridhara Chakravarthy. Steven Zimmerman and Alexander F. Schier are additional authors from Harvard University. Kourosh Salehi-Ashtiani and Marc Vidal are authors from Harvard Medical School. The work was supported by grants from the National Institutes of Health, the European Research Council, University College London, the High-Tech Fund of the Dana Farber Cancer Institute, the Ellison Foundation, the Edward Mallinckrodt, Jr. Foundation, the Rita Allen Foundation, and the Brain and Behavior Research Foundation.

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Kimm Fesenmaier
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A Gene That Helps Regulate Sleep
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By conducting a genetic screen in zebrafish, biologists have identified a gene that seems to serve as nature's stimulant.
Monday, February 29, 2016

Modeling molecules at the microscale

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