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

Geobiologist Honored by National Academy of Sciences

Dianne Newman, professor of biology and geobiology at Caltech and an investigator with the Howard Hughes Medical Institute, has been awarded the National Academy of Sciences (NAS) Award in Molecular Biology for her "discovery of microbial mechanisms underlying geologic processes." The award citation recognizes her for "launching the field of molecular geomicrobiology" and fostering greater awareness of the important roles microorganisms have played and continue to play in how Earth evolved.

"Trust me, no one was more shocked than I was by this news," says Newman. "It really honors the many the exceptional people who have come through my lab over the years, as well as the geobiology field more broadly. Geobiology is a venerable old field, which offers many fascinating and important problems that would benefit from the attention of individuals trained in mechanistic research. Hopefully this award will encourage more young people from molecular and cellular biology to enter the field."

Newman's research focuses on the relationship between microorganisms and geologic processes. She has demonstrated that some bacteria in iron-rich environments, such soils and sediments, can utilize extracellular iron as a dump site for excess electrons by generating extracellular electron shuttles, including a class of metabolites formerly considered to be redox-active antibiotics. Newman has also made contributions to our understanding of other microbial metabolic processes of geological significance, including how microbes respire using arsenate instead of oxygen, and how they perform photosynthesis using iron rather than water. In addition, she and her coworkers have studied the mechanisms by which certain microbes make stromatolites and magnetosomes, two types of structures that leave biosignatures in ancient rocks. Perhaps most importantly, her team has demonstrated the power of applying genetic analysis to diverse organisms from iron-rich environments, paving the way for others to do the same.

Newman is now hoping to bring tools commonly used in geochemistry to facilitate environmentally-informed studies of pathogens in chronic infections. For example, in collaboration with Caltech professor of geobiology Alex Sessions and researchers at Children's Hospital Los Angeles, Newman's group has characterized the composition and growth rate of pathogens in mucus collecting in the lungs of individuals with cystic fibrosis. Using this information, her lab is designing new experiments to reveal the survival mechanisms utilized by microorganisms—such as Pseudomonas aeruginosa, an opportunistic bacterium that colonizes the lungs of these patients—in this environment.

The NAS Award in Molecular Biology was first given in 1962. It is presented with a medal and a $25,000 prize. Newman will receive the award on May 1, 2016, during the National Academy of Sciences' annual meeting in Washington, D.C.

Previous recipients of the award include David Baltimore, Caltech President Emeritus and the Robert Andrews Millikan Professor of Biology.

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Dianne Newman has been awarded the National Academy of Sciences Award in Molecular Biology.

Delivering Genes Across the Blood-Brain Barrier

Caltech biologists have modified a harmless virus in such a way that it can successfully enter the adult mouse brain through the bloodstream and deliver genes to cells of the nervous system. The virus could help researchers map the intricacies of the brain and holds promise for the delivery of novel therapeutics to address diseases such as Alzheimer's and Huntington's. In addition, the screening approach the researchers developed to identify the virus could be used to make additional vectors capable of targeting cells in other organs.

"By figuring out a way to get genes across the blood-brain barrier, we are able to deliver them throughout the adult brain with high efficiency," says Ben Deverman, a senior research scientist at Caltech and lead author of a paper describing the work in the February 1 online publication of the journal Nature Biotechnology.

The blood-brain barrier allows the body to keep pathogens and potentially harmful chemicals circulating in the blood from entering the brain and spinal cord. The semi-permeable blockade, composed of tightly packed cells, is crucial for maintaining a controlled environment to allow the central nervous system to function properly. However, the barrier also makes it nearly impossible for many drugs and other molecules to be delivered to the brain via the bloodstream.

To sneak genes past the blood-brain barrier, the Caltech researchers used a new variant of a small, harmless virus called an adeno-associated virus (AAV). Over the past two decades, researchers have used various AAVs as vehicles to transport specific genes into the nuclei of cells; once there, the genes can be expressed, or translated, from DNA into proteins. In some applications, the AAVs carry functional copies of genes to replace mutated forms present in individuals with genetic diseases. In other applications, they are used to deliver genes that provide instructions for generating molecules such as antibodies or fluorescent proteins that help researchers study, identify, and track certain cells.

Largely because of the blood-brain barrier problem, scientists have had only limited success delivering AAVs and their genetic cargo to the central nervous system. In general, they have relied on surgical injections, which deliver high concentrations of the virus at the injection site but little to the outlying areas. Such injections are also quite invasive. "One has to drill a hole through skull, then pierce tissue with a needle to the injection site," explains Viviana Gradinaru (BS '05), assistant professor of biology and biological engineering at Caltech and senior author on the paper. "The deeper the injection, the higher the risk of hemorrhage. With systemic injection, using the bloodstream, none of that damage happens, and the delivery is more uniform."

In addition, Gradinaru notes, "many disorders are not tightly localized. Neurodegenerative disorders like Huntington's disease affect very large brain areas. Also, many complex behaviors are mediated by distributed interacting networks. Our ability to target those networks is key in terms of our efforts to understand what those pathways are doing and how to improve them when they are not working well."

In 2009, a group led by Brian Kaspar of Ohio State University published a paper, also in Nature Biotechnology, showing that an AAV strain called AAV9 injected into the bloodstream could make its way into the brain—but it was only efficient when used in neonatal, or infant, mice.

"The big challenge was how do we achieve the same efficiency in an adult," says Gradinaru.

Although one might like to design an AAV that is up to the task, the number of variables that dictate the behavior of any given virus, as well as the intricacies of the brain and its barrier, make that extremely challenging. Instead, the researchers developed a high-throughput selection assay, CREATE (Cre REcombinase-based AAV Targeted Evolution), that allowed them to test millions of viruses in vivo simultaneously and to identify those that were best at entering the brain and delivering genes to a specific class of brain cells known as astrocytes.

They started with the AAV9 virus and modified a gene fragment that codes for a small loop on the surface of the capsid—the protein shell of the virus that envelops all of the virus' genetic material. Using a common amplification technique, known as polymerase chain reaction (PCR), they created millions of viral variants. Each variant carried within it the genetic instructions to produce more capsids like itself.

Then they used their novel selection process to determine which variants most effectively delivered genes to astrocytes in the brain. Importantly, the new process relies on strategically positioning the gene encoding the capsid variants on the DNA strand between two short sequences of DNA, known as lox sites. These sites are recognized by an enzyme called Cre recombinase, which binds to them and inverts the genetic sequence between them. By injecting the modified viruses into transgenic mice that only express Cre recombinase in astrocytes, the researchers knew that any sequences flagged by the lox site inversion had successfully transferred their genetic cargo to the target cell type—here, astrocytes.

After one week, the researchers isolated DNA from brain and spinal cord tissue, and amplified the flagged sequences, thereby recovering only the variants that had entered astrocytes.

Next, they took those sequences and inserted them back into the modified viral genome to create a new library that could be injected into the same type of transgenic mice. After only two such rounds of injection and amplification, a handful of variants emerged as those that were best at crossing the blood-brain barrier and entering astrocytes.

"We went from millions of viruses to a handful of testable, potentially useful hits that we could go through systematically and see which ones emerged with desirable properties," says Gradinaru.

Through this selection process, the researchers identified a variant dubbed AAV-PHP.B as a top performer. They gave the virus its acronym in honor of the late Caltech biologist Paul H. Patterson because Deverman began this work in Patterson's group. "Paul had a commitment to understanding brain disorders, and he saw the value in pushing tool development," says Gradinaru, who also worked in Patterson's lab as an undergraduate student.

To test AAV-PHP.B, the researchers used it to deliver a gene that codes for a protein that glows green, making it easy to visualize which cells were expressing it. They injected the AAV-PHP.B or AAV9 (as a control) into different adult mice and after three weeks used the amount of green fluorescence to assess the efficacy with which the viruses entered the brain, the spinal cord, and the retina.

"We could see that AAV-PHP.B was expressed throughout the adult central nervous system with high efficiency in most cell types," says Gradinaru. Indeed, compared to AAV9, AAV-PHP.B delivers genes to the brain and spinal cord at least 40 times more efficiently.  

"What provides most of AAV-PHP.B's benefit is its increased ability to get through the vasculature into the brain," says Deverman. "Once there, many AAVs, including AAV9 are quite good at delivering genes to neurons and glia."

Gradinaru notes that since AAV-PHP.B is delivered through the bloodstream, it reaches other parts of the body. "Although in this study we were focused on the brain, we were also able to use whole-body tissue clearing to look at its biodistribution throughout the body," she says.

Whole-body tissue clearing by PARS CLARITY, a technique developed previously in the Gradinaru lab to make normally opaque mammalian tissues transparent, allows organs to be examined without the laborious task of making thin slide-mounted sections. Thus, tissue clearing allows researchers to more quickly screen the viral vectors for those that best target the cells and organs of interest.

"In this case, the priority was to express the gene in the brain, but we can see by using whole-body clearing that you can actually have expression in many other organs and even in the peripheral nerves," explains Gradinaru. "By making tissues transparent and looking through them, we can obtain more information about these viruses and identify targets that we might overlook otherwise."

The biologists conducted follow-up studies up to a year after the initial injections and found that the protein continued to be expressed efficiently. Such long-term expression is important for gene therapy studies in humans. 

In collaboration with colleagues from Stanford University, Deverman and Gradinaru also showed that AAV-PHP.B is better than AAV9 at delivering genes to human neurons and glia.

The researchers hope to begin testing AAV-PHP.B's ability to deliver potentially therapeutic genes in disease models. They are also working to further evolve the virus to make even better performing variants and to produce variants that target certain cell types with more specificity.

Deverman says that the CREATE system could indeed be applied to develop AAVs capable of delivering genes specifically to many different cell types. "There are hundreds of different Cre transgenic lines available," he says. "Researchers have put Cre recombinase under the control of gene regulatory elements so that it is only made in certain cell types. That means that regardless of whether your objective is to target liver cells or a particular type of neuron, you can almost always find a mouse that has Cre recombinase expressed in those cells."

"The CREATE system gave us a good hit early on, but we are excited about the future potential of using this approach to generate viruses that have very good cell-type specificity in different organisms, especially the less genetically tractable ones," says Gradinaru. "This is just the first step. We can take these tools and concepts in many exciting directions to further enhance this work, and we—with the Beckman Institute and collaborators—are ready to pursue those possibilities." 

The Beckman Institute at Caltech recently opened a resource center called CLOVER (CLARITY, Optogenetics, and Vector Engineering Research Center) to support such research efforts involving tissue clearing and imaging, optogenetic studies, and custom gene-delivery vehicle development. Deverman is the center's director, and Gradinaru is the principal investigator.

Additional Caltech authors on the paper, "Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain," are Sripriya Ravindra Kumar, Ken Y. Chan, Abhik Banerjee, Wei-Li Wu, and Bin Yang, as well as former Caltech students Piers L. Pravdo and Bryan P. Simpson. Nina Huber and Sergiu P. Pasca of Stanford University School of Medicine are also coauthors. The work was supported by funding from the Hereditary Disease Foundation and the Caltech-City of Hope Biomedical Initiative, a National Institutes of Health (NIH) Director's New Innovator Award, the NIH's National Institute of Aging and National Institute of Mental Health, the Beckman Institute, and the Gordon and Betty Moore Foundation.

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Delivering Genes Across the Blood-Brain Barrier
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Caltech biologists have developed a vector capable of noninvasive delivery of genetic cargo throughout the adult central nervous system.

Rosens Recharge Support for Bioengineering

Caltech board chair emeritus and longtime Compaq chairman Benjamin M. (Ben) Rosen (BS '54) and his wife, Donna, have made a bequest commitment to advance scientific exploration at the intersection of biology and engineering. It is anticipated that the couple's latest gift may double the endowment for the Donna and Benjamin M. Rosen Bioengineering Center.

Established in 2008 with $18 million from the Benjamin M. Rosen Family Foundation of New York, the Rosen Center has become a hub for research and educational initiatives that bring together applied physics, chemical engineering, synthetic biology, computer science, and more.

"Just as we had the digital revolution in the last century, we are having a biological sciences revolution in this century," Ben Rosen says. "And Caltech is the place to be."

Read more on the Caltech giving site.

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Rosens Recharge Support for Bioengineering
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Caltech board chair emeritus Ben Rosen (BS ’54) and his wife Donna have made a commitment to scientific exploration at the intersection of biology and engineering.
Friday, January 29, 2016
Center for Student Services 360 (Workshop Space) – Center for Student Services

Course Ombudsperson Training, Winter 2016

A Healthy Start

Science and medicine, it would seem, have always gone hand in hand. But for centuries, they were actually two very disparate fields. Identifying a need for "investigators who are well trained in both basic science and clinical research," the National Institutes of Health (NIH) created the Medical Scientist Training Program (MSTP) in 1964 to help streamline completion of dual medical and doctoral degrees. The purpose of developing this highly competitive MD/PhD program was to support "the training of students with outstanding credentials and potential who are motivated to undertake careers in biomedical research and academic medicine."

Recognizing Caltech's strength in the biological and chemical sciences, UCLA—which first established an MSTP in 1983—formed an affiliation with the Institute in 1997 to offer an average of two students the opportunity to perform graduate research at the partner school through the MSTP; PhD thesis work is done at Caltech for UCLA medical students, and when completed they return to UCLA to finish their MD studies.

The vast majority of alumni who have completed their postgraduate training are actively involved in biomedical research as physician-scientists at outstanding research institutions across the country. Although the MSTP represented the first formal affiliation between UCLA and Caltech, the success of the combined UCLA-Caltech MSTP spearheaded and served as a model for several other joint efforts that benefit from the complementary strengths of the two institutions, including the Specialized Training and Advanced Research (STAR) fellowship program for physician-scientists, and the Institute for Molecular Medicine.

A joint program with the University of Southern California soon followed. In 1998, the Kenneth T. and Eileen L. Norris Foundation awarded Caltech funding to support a joint MD/PhD program with the Keck School of Medicine of USC.

The grant established the Norris Foundation MD/PhD Scholars Fund, which supports Caltech PhD candidates from Keck. Administered by Caltech in cooperation with USC, the program accepts two students each year. As with the UCLA program, students spend their first two years in medical school, taking preclinical science courses, with summers spent at Caltech gaining exposure to the academic research environment. They then come to Caltech, spending three to five years on their PhDs before returning to their medical school for the final two clinical years.

The late Caltech biologist Paul Patterson, who passed away in 2014, was instrumental in developing the joint degree program. He believed that Caltech graduate students should also have an opportunity to explore their work in a clinical setting.

"Paul showed creativity both in curriculum development, in student mentoring, and in bringing the Caltech faculty together to support a program, which was in collaboration with another major institution," says Richard Bergman, director of the Cedars-Sinai Diabetes and Obesity Research Institute, who helped Patterson form the initial collaboration with USC. "His contributions in this regard educated several generations of students who, today, continue to make important contributions to medical science. This was a great legacy of Professor Patterson."

Additional funding for students in the MD/PhD programs has come from a provost-directed endowed fund called the W. R. Hearst Endowed Scholarship for MD/PhD Students; from the Lee-Ramo Life Sciences Fund; and through lab support for medical research from the W. M. Keck Foundation Fund for Discovery in Basic Medical Research. The Division of Biology and Biological Engineering also provides support to students and scholars who are headed for careers in medicine through an endowed fund from the Walter and Sylvia Treadway Foundation.

Since the start of the two MD/PhD programs, 64 students have been accepted to work toward dual degrees, and 40 have received PhDs from Caltech.

This story was reprinted from the Winter 2015 E&S magazine. See the full issue online.

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Explore the origins of Caltech's joint MD/PhD programs, which help students develop expertise in both basic science and clinical research.
Saturday, January 30, 2016
Beckman Institute Auditorium – Beckman Institute

Stem Cells, Gene Regulatory Networks and the Evolution of Vertebrates: A symposium recognizing the contributions of Marianne Bronner to our understanding of the neural crest and cranial placodes

Friday, January 29, 2016
Beckman Institute Auditorium – Beckman Institute

Stem Cells, Gene Regulatory Networks and the Evolution of Vertebrates: A symposium recognizing the contributions of Marianne Bronner to our understanding of the neural crest and cranial placodes

Identification Tags Define Neural Circuits

The human brain is composed of complex circuits of neurons, cells that are specialized to transmit information via electrochemical signals. Like the circuits in a computer, these neuronal circuits must be connected in particular ways to function properly. But with billions of neurons in a single human brain, how does a neuron make the right connections with the right cells?

Biologists have long searched for some kind of cellular "identification tags" that label which cells should form connections. Now, researchers from the laboratory of Caltech professor of biology Kai Zinn have identified molecules that act like identification tags on neurons in the fruit fly Drosophila. They discovered that proteins from two different molecular subfamilies, called Dpr and DIP proteins, bind together selectively. This binding can cause neurons that express Dpr proteins to form connections with neurons that express the corresponding DIP protein, playing an important role in directing the development of the neuromuscular and visual systems in growing Drosophila.

A paper detailing the findings is published in the December 17 issue of the journal Cell.

In 2013, a collaboration between Christopher Garcia's structural biology group at Stanford and the Zinn group at Caltech mapped the interactions between all 200 different Drosophila cell surface proteins. By separating the proteins from the cell and observing their interactions in a test tube, the group determined which proteins bind together. The group developed a complex model of interacting proteins they called the interactome. This work showed that a 21-member subfamily of "immunoglobulin superfamily" proteins, the Dprs, selectively bind to a 9-member subfamily called DIPs.

"Certain members of the Dprs and the DIPs match up and bind together—kind of like a lock and key—in a test tube," says Zinn. "We wanted to know if they would bind in vivo, in the Drosophila brain, and if that binding would then determine where synapses were formed."

A synapse is a junction where the wire-like axon of one neuron meets the branched dendrites of another. Information, in the form of chemical signals called neurotransmitters, is passed between neurons across these synapses. "We wanted to know if these interacting proteins on the surface of neuronal cells affected the way that the cells themselves interacted," says Robert Carrillo, a postdoctoral scholar in the Zinn group and co-first author on the new paper. "We showed that neural cells that expressed matching proteins often formed synapses with each other, and we theorized that the interaction between these molecules was driving the formation of synapses."

To test this theory, the Zinn group used the well-studied Drosophila visual system to determine the effects of these proteins on development. Neurons in the fly's eye send axons into layered structures in the visual part of the brain, which is known as the optic lobe. One of these structures, the medulla, is divided into ten layers, and each optic lobe neuron forms synapses within a specific subset of these layers. By removing certain DIP and Dpr proteins in the fly pupa, the researchers caused the axons to "overshoot" their target layers. Additionally, they observed developmental defects in the fly's neuromuscular system when removing the same proteins. Another paper in the same issue of Cell, from Larry Zipursky's group at UCLA, also found that expression of Dprs and DIPs correlates with the patterns of synaptic connectivity in the brain.

This finding helps to validate a theory proposed in the 1950s by the late Caltech professor and Nobel Laureate Roger Sperry. Experimenting mostly with fish and frog brains, Sperry discovered that he could manipulate or cut axons between neurons, and the cells would still re-form the right connections.

"Sperry hypothesized that individual neurons must carry some kind of identification tags, whose recognition is used to create the synaptic circuits of the brain," says Kaushiki Menon, a senior postdoctoral scholar in the Zinn group and a co-first author on the paper. "Our group has shown that the Drosophila Dpr and DIP proteins fit the definition of Sperry's proposed cellular identification tags."

Such tinkering with the brain's circuitry is possible because flies, unlike humans, have brains that are predominantly "hard-wired." "In mammals, the brain has a basic initial scaffold laid down by genetics, and then over time there is a lot of complicated experience-dependent rearrangement. Essentially, the brain can rewire itself through experience," Zinn says. "Fly brains can't do that."

While their findings are not immediately generalizable to mammals, Zinn and his group hope that they can provide a starting point to probe the structure of the human brain. "We hope that there might be protein networks that function similarly in humans, and these could be relevant to an understanding of how the scaffold of the human brain that exists at birth is assembled through genetics."

The paper is titled "Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins." In addition to Carrillo and Menon, structural biologist Engin Özkan at the University of Chicago is a  co-first author. The work was funded by the National Institutes of Health and the Howard Hughes Medical Institute.

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Lori Dajose
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Identification Tags Define Neural Circuits
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Biologists have identified a network of proteins that guides neural synapse formation in Drosophila brains.

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