Genetically Engineered Antibodies Show Enhanced HIV-Fighting Abilities

Capitalizing on a new insight into HIV's strategy for evading antibodies—proteins produced by the immune system to identify and wipe out invading objects such as viruses—Caltech researchers have developed antibody-based molecules that are more than 100 times better than our bodies' own defenses at binding to and neutralizing HIV, when tested in vitro. The work suggests a novel approach that could be used to engineer more effective HIV-fighting drugs.

"Based on the work that we have done, we now think we know how to make a really potent therapeutic that would not only work at relatively low concentrations but would also force the virus to mutate along pathways that would make it less fit and therefore more susceptible to elimination," says Pamela Bjorkman, the Max Delbrück Professor of Biology and an investigator with the Howard Hughes Medical Institute. "If you were able to give this to someone who already had HIV, you might even be able to clear the infection."

The researchers describe the work in the January 29 issue of Cell. Rachel Galimidi, a graduate student in Bjorkman's lab at Caltech, is lead author on the paper.

The researchers hypothesized that one of the reasons the immune system is less effective against HIV than other viruses involves the small number and low density of spikes on HIV's surface. These spikes, each one a cluster of three protein subunits, stick up from the surface of the virus and are the targets of antibodies that neutralize HIV. While most viruses are covered with hundreds of these spikes, HIV has only 10 to 20, making the average distance between the spikes quite long.

That distance is important with respect to the mechanism that naturally occurring antibodies use to capture their viral targets. Antibodies are Y-shaped proteins that evolved to grab onto their targets with both "arms." However, if the spikes are few and far between—as is the case with HIV—it is likely that an antibody will bind with only one arm, making its connection to the virus weaker (and easier for a mutation of the spike to render the antibody ineffective).

To test their hypothesis, Bjorkman's group genetically engineered antibody-based molecules that can bind with both arms to a single spike. They started with the virus-binding parts, or Fabs, of broadly neutralizing antibodies—proteins produced naturally by a small percentage of HIV-positive individuals that are able to fight multiple strains of HIV until the virus mutates. When given in combination, these antibodies are quite effective. Rather than making Y-shaped antibodies, the Caltech group simply connected two Fabs—often from different antibodies, to mimic combination therapies—with different lengths of spacers composed of DNA.

Why DNA? In order to engineer antibodies that could latch onto a spike twice, they needed to know which Fabs to use and how long to make the connection between them so that both could readily bind to a single spike. Previously, various members of Bjorkman's group had tried to make educated guesses based on what is known of the viral spike structure, but the large number of possible variations in terms of which Fabs to use and how far apart they should be, made the problem intractable.

In the new work, Bjorkman and Galimidi struck upon the idea of using DNA as a "molecular ruler." It is well known that each base pair in double-stranded DNA is separated by 3.4 angstroms. Therefore, by incorporating varying lengths of DNA between two Fabs, they could systematically test for the best neutralizer and then derive the distance between the Fabs from the length of the DNA. They also tested different combinations of Fabs from various antibodies—sometimes incorporating two different Fabs, sometimes using two of the same.

"Most of these didn't work at all," says Bjorkman, which was reassuring because it suggested that any improvements the researchers saw were not just created by an artifact, such as the addition of DNA.

But some of the fabricated molecules worked very well. The researchers found that the molecules that combined Fabs from two different antibodies performed the best, showing an improvement of 10 to 1,000 times in their ability to neutralize HIV, as compared to naturally occurring antibodies. Depending on the Fabs used, the optimal length for the DNA linker was between 40 and 62 base pairs (corresponding to 13 and 21 nanometers, respectively).

Taking this finding to the next level in the most successful of these new molecules, the researchers replaced the piece of DNA with a protein linker of roughly the same length composed of 12 copies of a protein called tetratricopeptide repeat. The end product was an all-protein antibody-based reagent designed to bind with both Fabs to a single HIV spike.

"That one also worked, showing more than 30-fold average increased potency compared with the parental antibodies," says Bjorkman. "That is proof of principle that this can be done using protein-based reagents."

The greater potency suggests that a reagent made of these antibody-based molecules could work at lower concentrations, making a potential therapeutic less expensive and decreasing the risk of adverse reactions in patients.

"I think that our work sheds light on the potential therapeutic strategies that biotech companies should be using—and that we will be using—in order to make a better antibody reagent to combat HIV," says Galimidi. "A lot of companies discount antibody reagents because of the virus's ability to evade antibody pressure, focusing instead on small molecules as drug therapies. Our new reagents illustrate a way to get around that."

The Caltech team is currently working to produce larger quantities of the new reagents so that they can test them in humanized mice—specialized mice carrying human immune cells that, unlike most mice, are sensitive to HIV.

Along with Galimidi and Bjorkman, additional Caltech authors on the paper, "Intra-Spike Crosslinking Overcomes Antibody Evasion by HIV-1," include Maria Politzer, a lab assistant; and Anthony West, a senior research specialist. Joshua Klein, a former Caltech graduate student (PhD '09), and Shiyu Bai, a former technician in the Bjorkman lab, also contributed to the work; they are currently at Google and Case Western Reserve University School of Medicine, respectively. Michael Seaman of Beth Israel Deaconess Medical Center and Michel Nussenzweig of the Rockefeller University in New York are also coauthors. The work was supported by the National Institutes of Health through a Director's Pioneer Award and a grant from the HIV Vaccine Research and Design Program, as well as grants from the Collaboration for AIDS Vaccine Discovery and the Bill and Melinda Gates Foundation. Nussenzweig is also an investigator with the Howard Hughes Medical Institute.

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Why Do We Feel Thirst? An Interview with Yuki Oka

To fight dehydration on a hot summer day, you instinctively crave the relief provided by a tall glass of water. But how does your brain sense the need for water, generate the sensation of thirst, and then ultimately turn that signal into a behavioral trigger that leads you to drink water? That's what Yuki Oka, a new assistant professor of biology at Caltech, wants to find out.

Oka's research focuses on the study of how the brain and body work together to maintain a healthy ratio of salt to water as part of a delicate form of biological balance called homeostasis.

Recently, Oka came to Caltech from Columbia University. We spoke with him about his work, his interests outside of the lab, and why he's excited to be joining the faculty at Caltech.

 

Can you tell us a bit more about your research?

The goal of my research is to understand the mechanisms by which the brain and body cooperate to maintain our internal environment's stability, which is called homeostasis. I'm especially focusing on fluid homeostasis, the fundamental mechanism that regulates the balance of water and salt. When water or salt are depleted in the body, the brain generates a signal that causes either a thirst or a salt craving. And that craving then drives animals to either drink water or eat something salty.

I'd like to know how our brain generates such a specific motivation simply by sensing internal state, and then how that motivation—which is really just neural activity in the brain—goes on to control the behavior.

 

Why did you choose to study thirst?

After finishing my Ph.D. in Japan, I came to Columbia University where I worked on salt sensing mechanisms in the mammalian taste system. We found that the peripheral taste system has a key function for salt homeostasis in the body by regulating our salt intake behavior. But of course, the peripheral sensor does not work by itself.  It requires a controller, the brain, which uses information from the sensor. So I decided to move on to explore the function of the brain; the real driver of our behaviors.

I was fascinated by thirst because the behavior it generates is very robust and stereotyped across various species. If an animal feels thirst, the behavioral output is simply to drink water. On the other hand, if the brain triggers salt appetite, then the animal specifically looks for salt—nothing else. These direct causal relations make it an ideal system to study the link between the neural circuit and the behavior.

 

You recently published a paper on this work in the journal Nature. Could you tell us about those findings?

In the paper, we linked specific neural populations in the brain to water drinking behavior. Previous work from other labs suggested that thirst may stem from a part of the brain called the hypothalamus, so we wanted to identify which groups of neurons in the hypothalamus control thirst. Using a technique called optogenetics that can manipulate neural activities with light, we found two distinct populations of neurons that control thirst in two opposite directions. When we activated one of those two populations, it evoked an intense drinking behavior even in fully water-satiated animals. In contrast, activation of a second population drastically suppressed drinking, even in highly water-deprived thirsty animals.  In other words, we could artificially create or erase the desire for drinking water.

Our findings suggest that there is an innate brain circuit that can turn an animal's water-drinking behavior on and off, and that this circuit likely functions as a center for thirst control in the mammalian brain. This work was performed with support from Howard Hughes Medical Institute and National Institutes of Health [for Charles S. Zuker at Columbia University, Oka's former advisor].

 

You use a mouse model to study thirst, but does this work have applications for humans?

There are many fluid homeostasis-associated conditions; one example is dehydration. We cannot specifically say a direct application for humans since our studies are focused on basic research. But if the same mechanisms and circuits exist in mice and humans, our studies will provide important insights into human physiologies and conditions.

 

Where did you grow up—and what started your initial interest in science?

I grew up in Japan, close to Tokyo, but not really in the center of the city. It was a nice combination between the big city and nature. There was a big park close to my house and when I was a child, I went there every day and observed plants and animals. That's pretty much how I spent my childhood. My parents are not scientists—neither of them, actually. It was just my innate interest in nature that made me want to be a scientist.

 

What drew you to Caltech?

I'm really excited about the environment here and the great climate. That's actually not trivial; I think the climate really does affect the people. For example, if you compare Southern California to New York, it's just a totally different character. I came here for a visit last January, and although it was my first time at Caltech I kind of felt a bond. I hadn't even received an offer yet, but I just intuitively thought, "This is probably the place for me."

I'm also looking forward to talking to my colleagues here who use fMRI for human behavioral research. One great advantage about using human subjects in behavioral studies is that they can report back to you about how they feel. There are certainly advantages of using an animal model, like mice. But they cannot report back. We just observe their behavior and say, "They are drinking water, so they must be thirsty." But that is totally different than someone telling you, "I feel thirsty." I believe that combining advantages of animal and human studies should allow us to address important questions about brain functions.

 

Do you have any hobbies?

I play basketball in my spare time, but my major hobby is collecting fossils. I have some trilobites and, actually, I have a complete set of bones from a type of herbivorous dinosaur. It is being shipped from New York right now and I may put it in my new office.

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How Do You Make a Greasy Protein?

Watson Lecture Preview

Every cell is encapsulated and protected by a thin membrane made of greasy molecules called lipids. Assemblies of equally greasy protein molecules span the membrane, forming passageways that control the flow of signaling molecules that, in turn, direct the cell's activities. Because of these proteins' key role in cell-to-cell communication, they have become a prime target for drug design. Professor of Biochemistry Bil Clemons is among those working out the structures of these proteins and, more fundamentally, the biological processes behind them. Clemons will discuss how cells assemble these proteins, and how they deliver them to the membrane, at 8 p.m. on Wednesday, January 7, in Caltech's Beckman Auditorium. Admission is free.

 

Q: What do you do?

A: I am nominally a structural biologist, but I'm really a crystallographer. We purify a protein in solution and then try to crystallize it, which is really, really hard. When we succeed, we make X-ray diffraction patterns of the crystals and work backwards from those patterns to calculate the precise position of every atom. This allows us to make a blueprint for the molecule, and the blueprint helps us understand how the molecule does what it does. That's my group's real interest—figuring out the biological mechanisms that underlie how a protein works. We want to understand, on a molecular level, the processes by which these proteins are targeted and inserted into the membrane.

Proteins are long chains of amino acids that assume very specific three-dimensional shapes, or conformations. The proteins we work on contain hundreds of amino acids and thousands of individual atoms. These proteins interact with other molecules as they do their jobs. When they do, their conformations change, so a large part of our work is trying to understand all these different interactions and motions.

A crystal contains millions of copies of the same molecule held in exactly one conformation, so in that sense, a crystal structure is just one snapshot of a series of biological motions. Eventually we'd like to make movies of all the conformational changes that occur during these interactions—or at least render the important frames. It's almost like producing a cheap cartoon, where the lead animator draws a few key cels, and the rest is filled in later.

 

Q: What do you get from a crystal structure?

A: We get the first glimpse of how something works. Every crystal structure provides a huge amount of information. The beauty of structural biology is that we get to be the first people to peek under the hood of a protein and draw a three-dimensional map of what we see. Science is vast, and most people work in very narrow fields, doing mechanistic studies and drug discovery and all sorts of things. Structural biologists create the platform for everyone else's studies.

 

Q: How did you get into this line of work?

A: Well, I'd like to say it was a series of happy accidents. I've always been passionate about science. In my heart, I think I was born a scientist. I always wanted to know how everything worked, and biochemistry fascinated me. There was so much complexity—so many ways to ask questions.

At Virginia Tech, I was lucky enough to have an undergraduate adviser, Walt Niehaus, who encouraged me to do research in his group. There was really no looking back after that. I just thought, "Wow. This is really fun. I like doing this." Meanwhile, I was paying my way through school. My senior year I was the student manager of one of the food-service facilities. I was working nearly 40 hours a week managing 40 employees plus spending another 20 hours in the lab and 20 hours in school. I wasn't able to look past that to what my future might be, but Walt pushed me to apply for grad school. It was eye-opening the first time he suggested I could do this for a living.

Walt's research was in basic biochemistry. There weren't any structural biologists at Virginia Tech at the time, but the Howard Hughes Medical Institute sent us a booklet with stereo pictures of protein structures. I thought, "You've got to be kidding me. We can look at these things in 3-D?" It blew my mind. So I went to grad school at the University of Utah to be a crystallographer, and I earned my PhD working on the molecular machinery responsible for making proteins. Then I did my postdoctoral work at Harvard Med, trying to understand the complex process of getting greasy membrane proteins into cell membranes. We solved the structure of an important piece of the puzzle there, and now that I'm at Caltech, which has major strengths in X-ray crystallography, we're filling in the details of the bigger picture.

 

 

Named for the late Caltech professor Earnest C. Watson, who founded the series in 1922, the Watson Lectures present Caltech and JPL researchers describing their work to the public. Many past Watson Lectures are available online at Caltech's iTunes U site.

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Improving The View Through Tissues and Organs

On Saturday, October 18, hundreds of undergraduate students will share the results of their projects during SURF Seminar Day. The event, which is open to the public, is an opportunity for students to discuss and explain their research to individuals with a wide-range of expertise and interests.

This summer, several undergraduate students at Caltech had the opportunity to help optimize a promising technique that can make tissues and organs—even entire organisms—transparent for study. As part of the Summer Undergraduate Research Fellowship (SURF) program, these students worked in the lab of Assistant Professor of Biology Viviana Gradinaru, where researchers are developing such so-called clearing techniques that make it possible to peer straight through normally opaque tissues rather than seeing them only as thinly sectioned slices that have been pieced back together.

Gradinaru's group recently published a paper in the journal Cell describing a new approach to tissue clearing. The method they have created builds on a technique called CLARITY that Gradinaru helped develop while she was a research associate at Stanford. CLARITY allowed researchers to, for the first time, create a transparent whole-brain specimen that could then be imaged with its structural and genetic information intact.

CLARITY was specifically developed for studying the brain. But the new approach developed in Gradinaru's lab, which the team has dubbed PARS (perfusion-assisted agent release in situ), can also clear other organs, such as the kidney, as well as tissue samples, such as tumor biopsies. It can even be applied to entire organisms.

Like CLARITY, PARS involves removing the light-scattering lipids in the tissue to make samples transparent without losing the structural integrity that lipids typically provide. First the sample is infused with acrylamide monomers that are then polymerized into a hydrogel that provides structural support. Next, this tissue–hydrogel hybrid is immersed in a detergent that removes the lipids. Then the sample can be stained, often with antibodies that specifically mark cells of interest, and then immersed in RIMS (refractive index matching solution) for imaging using various optical techniques such as confocal or lightsheet microscopy.

Over the summer, Sam Wie, a junior biology major at Caltech, spent 10 weeks in the Gradinaru lab working to find a polymer that would perform better than acrylamide, which has been used in the CLARITY hydrogel. "One of the limitations of CLARITY is that when you put the hydrogel tissue into the detergent, the higher solute concentration in the tissue causes liquid to rush into the cell. That causes the sample to swell, which could potentially damage the structure of the tissue," Wie explains. "So I tried different polymers to try to limit that swelling."

Wie was able to identify a polymer that produces, over a similar amount of time, about one-sixth of the swelling in the tissue.

"The SURF experience has been very rewarding," Wie says. "I've learned a lot of new techniques, and it's really exciting to be part of, and to try to improve, CLARITY, a method that will probably change the way that we image tissues from now on."

At another bench in Gradinaru's lab, sophomore bioengineering major Andy Kim spent the summer focusing on a different aspect of the PARS technique. While antibodies have been the most common markers used to tag cells of interest within cleared tissues, they are too large for some studies—for example, those that aim to image deeper parts of the brain, requiring them to cross the blood–brain barrier. Kim's project involved identifying smaller proteins, such as nanobodies, which target and bind to specific parts of proteins in tissues.

"While PARS is a huge improvement over CLARITY, using antibodies to stain is very expensive," Kim says. "However, some of these nanobodies can be produced easily, so if we can get them to work, it would not only help image the interior of the brain, it would also be a lot less costly."

During his SURF, Kim worked with others in the lab to identify about 30 of these smaller candidate binding proteins and tested them on PARS-cleared samples.

While Wie and Kim worked on improving the PARS technique itself, Donghun Ryu, a third SURFer in Gradinaru's lab, investigated different methods for imaging the cleared samples. Ryu is a senior electrical engineering and computer science major at the Gwangju Institute of Science and Technology (GIST) in the Republic of Korea.

Last summer Ryu completed a SURF as part of the Caltech–GIST Summer Undergraduate Research Exchange Program in the lab of Changhuei Yang, professor of electrical engineering, bioengineering, and medical engineering at Caltech. While completing that project, Ryu became interested in optogenetics, the use of light to control genes. Since optogenetics is one of Gradinaru's specialties, Yang suggested that he try a SURF in Gradinaru's lab.

This summer, Ryu was able to work with both Yang and Gradinaru, investigating a technique called Talbot microscopy to see whether it would be better for imaging thick, cleared tissues than more common techniques. Ryu was able to work on the optical system in Yang's lab while testing the samples cleared in Gradinaru's lab.

"It was a wonderful experience," Ryu says. "It was special to have the opportunity to work for two labs this summer. I remember one day when I had a meeting with both Professor Yang and Professor Gradinaru; it was really amazing to get to meet with two Caltech professors."

Gradinaru says that the SURF projects provided a learning opportunity not only for the participating students but also for her lab. "For example," she says, "Ryu strengthened the collaboration that we have with the Yang group for the BRAIN Initiative. And my lab members benefited from the chance to serve as mentors—to see what works and what can be improved when transferring scientific knowledge. These are very important skills in addition to the experimental know-how that they master."  

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Sensors to Simplify Diabetes Management

For many patients diagnosed with diabetes, treating the disease can mean a burdensome and uncomfortable lifelong routine of monitoring blood sugar levels and injecting the insulin that their bodies don't naturally produce. But, as part of their Summer Undergraduate Research Fellowship (SURF) projects at Caltech, several engineering students have contributed to the development of tiny biosensors that could one day eliminate the need for these manual blood sugar tests.

Because certain patients with diabetes are unable to make their own insulin—a hormone that helps transfer glucose, or sugar, from the blood into muscle and other tissues—they need to monitor frequently their blood glucose, manually injecting insulin when sugar levels surge after a meal. Most glucose monitors require that patients prick their fingertips to collect a drop of blood, sometimes up to 10 times a day for the rest of their lives.

In their SURF projects, the students, all from Caltech's Division of Engineering and Applied Science, looked for different ways to do these same tests but painlessly and automatically.

Mehmet SencanSenior applied physics major Mehmet Sencan has approached the problem with a tiny chip that can be implanted under the skin. The sensor, a square just 1.4 millimeters on each side, is designed to detect glucose levels from the interstitial fluid (fluid found in the spaces between cells) that is just under the skin. The glucose levels in this fluid directly relate to the blood glucose concentration.

Sencan has been involved in optimizing the electrochemical method that the chip will use to detect glucose levels. Much like a traditional finger-stick glucose meter, the chip uses glucose oxidase, an enzyme that reacts in the presence of glucose, to create an electrical current. Higher levels of glucose result in a stronger current, allowing the device to measure glucose levels based on the charge that passes through the fluid.

Once the glucose level is detected, the information is wirelessly transmitted via a radio wave frequency to a reader that uses the same frequency to power the device itself. Ultimately an external display will let the patient know if their levels are within range.

Sencan, who works in the laboratory of Axel Scherer, the Bernard Neches Professor of Electrical Engineering, Applied Physics, and Physics, and who is co-mentored by postdoctoral researcher Muhammad Mujeeb-U-Rahman, started this project three years ago during his very first SURF.

"When I started, we were just thinking about what kind of chemistry the sensor would use, and now we have a sensor that is actually designed to do that," he says. Over the summer, he implanted the sensors in rat models, and he will continue the study over the fall and spring terms using both rat and mouse models—a first step in determining if the design is a clinically viable option.

Sith DomrongkitchaipornJunior electrical engineering major Sith Domrongkitchaiporn from the Scherer laboratory, also co-mentored by Mujeeb-U-Rahman, took a different approach to glucose detection, making tiny biosensors that are inconspicuously wearable on the surface of a contact lens. "It's an interesting concept because instead of having to do a procedure to place something under the skin, you can use a less invasive method, placing a sensor on the eye to get the same information," he says.

He used the method optimized by Mehmet to determine blood glucose levels from interstitial fluid and adapted the chemistry to measure glucose in the eyes' tears. This summer, he will be attempting to fabricate the lens itself and improve upon the process whereby radio waves are used to power the sensor and then transmit data from the sensor to an external computer.

Jennifer Chih-Wen LinSURF student and sophomore electrical engineering major Jennifer Chih-Wen Lin wanted to incorporate a different kind of glucose sensor into a contact lens. "The concept—determining glucose readings from tears—is very similar to Sith's, but the method is very different," she says.

Instead of determining the glucose level based on the amount of electrical current that passes through a sample, Lin, who works in the laboratory of Hyuck Choo, assistant professor of electrical engineering, worked on a sensor that detects glucose levels from the interaction between light and molecules.

In her SURF project, she began optimizing the characterization of glucose molecules in a sample of glucose solution using a technique called Raman spectroscopy. When molecules encounter light, they vibrate differently based on their symmetry and the types of bonds that hold their atoms together. This vibrational information provides a unique fingerprint for each type of molecule, which is represented as peaks on the Raman spectrum—and the intensity of these peaks correlates to the concentration of that molecule within the sample.

"This step is important because once I can determine the relationship between peak intensities and glucose concentrations, our sensor can just compare that known spectrum to the reading from a sample of tears to determine the amount of glucose in the sample," she says.

Lin's project is in the very beginning stages, but if it is successful, it could provide a more accurate glucose measurement, and from a smaller volume of liquid, than is possible with the finger-stick method. Perhaps more importantly for patients, it can provide that measurement painlessly.

Sophia ChenAlso in Choo's laboratory, sophomore electrical engineering major Sophia Chen's SURF project involves a new way to power devices like these tiny sensors and other medical implants, using the vibrations from a patient's vocal cords. These vibrations produce the sound of our voice, and also create vibrations in the skull.

"We're using these devices called energy harvesters that can extract energy from vibrations at specific frequencies. When the vibrations go from the vocal folds to the skull, a structure in the energy harvester vibrates at the same frequency, generating energy—energy that can be used to power batteries or charge things," Chen says.

Chen's goal is to determine the frequency of these vibrations—and if the energy that they produce is actually enough to power a tiny device. The hope is that one day these vibrations could power, or at least supplement the power of, medical devices that need to be implanted near the head and that presently run on batteries with finite lifetimes.

Chen and the other students acknowledge that health-monitoring sensors powered by the human body might be years away from entering the clinic. However, this opportunity to apply classroom knowledge to a real-life challenge—such as diabetes treatment—is an important part of their training as tomorrow's scientists and engineers.

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Orchestrating the Healing Process in a Damaged Cornea

It is safe to say that the eye is an amazing biological system. One reason is its keratocyte cells—specialized cells that make up the bulk of the cornea. Unlike most of the other cells in our body, those in the cornea are transparent, making sight possible. Should something happen to make the cornea opaque, blindness results.

Sadly, injuries to the cornea do occur, sometimes in the simplest of ways, such as getting sand in one's eyes and scratching the cornea. The scar tissue that then grows to heal the cornea may have the unwanted side effect of being opaque. This does not happen, however, if the cornea and the tissue around it heal in a very orderly fashion. The question, then: Is it possible to encourage this orderly healing after an injury, thus preserving vision?

Professor of Chemical Engineering Julia Kornfield and graduate student Amy Fu are very much hoping that this is the case. To find out, they have assigned a few students, including Caltech senior and recent SURF fellow Jacqueline Masehi-Lano, to experiment with various growth factors that might inhibit the formation of scar tissue and promote orderly wound healing.

"We chose three growth factors to test because Amy Fu and I read several papers on growth factors that have been able to suppress some types of scar tissue," Masehi-Lano says. "In particular, we want to inhibit the formation of alpha smooth muscle actin, the type of stress fiber that creates opaque scars over corneal wounds. So far, the experiments I've done with cell cultures have worked pretty well, so it looks promising."

Eventually, the researchers hope to encapsulate the growth factors in a hydrogel that is reminiscent of the native cornea. "Our hydrogel starts out as a liquid and gels in situ on the eye," explains Masehi-Lano.

Masehi-Lano is enthusiastic about her experience with the SURF program. This past summer was her second in Kornfield's lab, and last year she was a recipient of an Amgen scholarship. "I'm really grateful that my mentor and my co-mentor have entrusted me with my own project and have allowed me to conduct my own experiments. And since it was my second summer in this lab, I was able to take up a leadership role by training a new SURF student," she says. "For me, SURF has gone beyond research. I've been able to improve my ability to present my research to the general public, which I think is extremely important." Indeed, Masehi-Lano was awarded the Caltech Doris S. Perpall SURF Speaking Competition for delivering the most outstanding oral research presentation.

Masehi-Lano plans to continue in bioengineering and is contemplating an MD/PhD program. "I've always been interested in the medical field, and though I'm committed to doing research," she explains, "I'd like to be able to do clinical trials and directly apply new medical technologies to people."

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A New Way to Prevent the Spread of Devastating Diseases

For decades, researchers have tried to develop broadly effective vaccines to prevent the spread of illnesses such as HIV, malaria, and tuberculosis. While limited progress has been made along these lines, there are still no licensed vaccinations available that can protect most people from these devastating diseases.

So what are immunologists to do when vaccines just aren't working?

At Caltech, Nobel Laureate David Baltimore and his colleagues have approached the problem in a different way. Whereas vaccines introduce substances such as antigens into the body hoping to illicit an appropriate immune response—the generation of either antibodies that might block an infection or T cells capable of attacking infected cells—the Caltech team thought: Why not provide the body with step-by-step instructions for producing specific antibodies that have been shown to neutralize a particular disease?

The method they developed—originally to trigger an immune response to HIV—is called vectored immunoprophylaxis, or VIP. The technique was so successful that it has since been applied to a number of other infectious diseases, including influenza, malaria, and hepatitis C.

"It is enormously gratifying to us that this technique can have potentially widespread use for the most difficult diseases that are faced particularly by the less developed world," says Baltimore, president emeritus and the Robert Andrews Millikan Professor of Biology at Caltech.

VIP relies on the prior identification of one or more antibodies that are able to prevent infection in laboratory tests by a wide range of isolated samples of a particular pathogen. Once that has been done, researchers can incorporate the genes that encode those antibodies into an adeno-associated virus (AAV), a small, harmless virus that has been useful in gene-therapy trials. When the AAV is injected into muscle tissue, the genes instruct the muscle tissue to generate the specified antibodies, which can then enter the circulation and protect against infection.

In 2011, the Baltimore group reported in Nature that they had used the technique to deliver antibodies that effectively protected mice from HIV infection. Alejandro Balazs was lead author on that paper and was a postdoctoral scholar in the Baltimore lab at the time.

"We expected that at some dose, the antibodies would fail to protect the mice, but it never did—even when we gave mice 100 times more HIV than would be needed to infect seven out of eight mice," said Balazs, now at the Ragon Institute of MGH, MIT and Harvard. "All of the exposures in this work were significantly larger than a human being would be likely to encounter."

At the time, the researchers noted that the leap from mice to humans is large but said they were encouraged by the high levels of antibodies the mice were able to produce after a single injection and how effectively the mice were protected from HIV infection for months on end. Baltimore's team is now working with a manufacturer to produce the materials needed for human clinical trials that will be conducted by the Vaccine Research Center at the National Institutes of Health.

Moving on from HIV, the Baltimore lab's next goal was protection against influenza A. Although reasonably effective influenza vaccines exist, each year more than 20,000 deaths, on average, are the result of seasonal flu epidemics in the United States. We are encouraged to get flu shots every fall because the influenza virus is something of a moving target—it evolves to avoid resistance. There are also many different strains of influenza A (e.g. H1N1 and H3N2), each incorporating a different combination of the various forms of the proteins hemagglutinin (H) and neuraminidase (N). To chase this target, the vaccine is reformulated each year, but sometimes it fails to prevent the spread of the strains that are prevalent that year.

But about five years ago, researchers began identifying a new class of anti-influenza antibodies that are able to prevent infection by many, many strains of the virus. Instead of binding to the head of the influenza virus, as most flu-fighting antibodies do, these new antibodies target the stalk that holds up the head. And while the head is highly adaptable—meaning that even when mutations occur there, the virus can often remain functional—the stalk must basically remain the same in order for the virus to survive. So these stalk antibodies are very hard for the virus to mutate against.

In 2013, the Baltimore group stitched the genes for two of these new antibodies into an AAV and showed that mice injected with the vector were protected against multiple flu strains, including all H1, H2, and H5 influenza strains tested. This was even true of older mice and those without a properly functioning immune system—a particularly important finding considering that most deaths from the flu occur in the elderly and immunocompromised populations. The group reported its results in the journal Nature Biotechnology.

"We have shown that we can protect mice completely against flu using a kind of antibody that doesn't need to be changed every year," says Baltimore. "It is important to note that this has not been tested in humans, so we do not yet know what concentration of antibody can be produced by VIP in humans. However, if it works as well as it does in mice, VIP may provide a plausible approach to protect even the most vulnerable patients against epidemic and pandemic influenza."

Now that the Baltimore lab has shown VIP to be so effective, other groups from around the country have adopted the Caltech-developed technique to try to ward off malaria, hepatitis C, and tuberculosis.

In August, a team led by Johns Hopkins Bloomberg School of Public Health reported in the Proceedings of the National Academy of Sciences (PNAS) that as many as 70 percent of mice that they had injected by the VIP procedure were protected from infection with malaria by Plasmodium falciparum, the parasite that carries the most lethal of the four types of the disease. A subset of mice in the study produced particularly high levels of the disease-fighting antibodies. In those mice, the immunization was 100 percent effective.

"This is also just a first-generation antibody," says Baltimore, who was a coauthor on the PNAS study. "Knowing now that you can get this kind of protection, it's worth trying to get much better antibodies, and I trust that people in the malaria field will do that."

Most recently, a group led by researchers from The Rockefeller University showed that three hepatitis-C-fighting antibodies delivered using VIP were able to protect mice efficiently from the virus. The results were published in the September 17 issue of the journal Science Translational Medicine. The researchers also found that the treatment was able to temporarily clear the virus from mice that had already been infected. Additional work is needed to determine how to prevent the disease from relapsing. Interestingly, though, the work suggests that the antibodies that are effective against hepatitis C, once it has taken root in the liver, may work by protecting uninfected liver cells from infection while allowing already infected cells to be cleared from the body.    

An additional project is currently evaluating the use of VIP for the prevention of tuberculosis—a particular challenge given the lack of proven tuberculosis-neutralizing antibodies.

"When we started this work, we imagined that it might be possible to use VIP to fight other diseases, so it has been very exciting to see other groups adopting the technique for that purpose," Baltimore says. "If we can get positive clinical results in humans with HIV, we think that would really encourage people to think about using VIP for these other diseases."

Baltimore's work is supported by funding from the National Institute of Allergy and Infectious Disease, the Bill and Melinda Gates Foundation, the Caltech-UCLA Joint Center for Translational Medicine, and a Caltech Translational Innovation Partnership Award.

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Sensing Neuronal Activity With Light

For years, neuroscientists have been trying to develop tools that would allow them to clearly view the brain's circuitry in action—from the first moment a neuron fires to the resulting behavior in a whole organism. To get this complete picture, neuroscientists are working to develop a range of new tools to study the brain. Researchers at Caltech have developed one such tool that provides a new way of mapping neural networks in a living organism.

The work—a collaboration between Viviana Gradinaru (BS '05), assistant professor of biology and biological engineering, and Frances Arnold, the Dick and Barbara Dickinson Professor of Chemical Engineering, Bioengineering and Biochemistry—was described in two separate papers published this month.

When a neuron is at rest, channels and pumps in the cell membrane maintain a cell-specific balance of positively and negatively charged ions within and outside of the cell resulting in a steady membrane voltage called the cell's resting potential. However, if a stimulus is detected—for example, a scent or a sound—ions flood through newly open channels causing a change in membrane voltage. This voltage change is often manifested as an action potential—the neuronal impulse that sets circuit activity into motion.

The tool developed by Gradinaru and Arnold detects and serves as a marker of these voltage changes.

"Our overarching goal for this tool was to achieve sensing of neuronal activity with light rather than traditional electrophysiology, but this goal had a few prerequisites," Gradinaru says. "The sensor had to be fast, since action potentials happen in just milliseconds. Also, the sensor had to be very bright so that the signal could be detected with existing microscopy setups. And you need to be able to simultaneously study the multiple neurons that make up a neural network."

The researchers began by optimizing Archaerhodopsin (Arch), a light-sensitive protein from bacteria. In nature, opsins like Arch detect sunlight and initiate the microbes' movement toward the light so that they can begin photosynthesis. However, researchers can also exploit the light-responsive qualities of opsins for a neuroscience method called optogenetics—in which an organism's neurons are genetically modified to express these microbial opsins. Then, by simply shining a light on the modified neurons, the researchers can control the activity of the cells as well as their associated behaviors in the organism.

Gradinaru had previously engineered Arch for better tolerance and performance in mammalian cells as a traditional optogenetic tool used to control an organism's behavior with light. When the modified neurons are exposed to green light, Arch acts as an inhibitor, controlling neuronal activity—and thus the associated behaviors—by preventing the neurons from firing.

However, Gradinaru and Arnold were most interested in another property of Arch: when exposed to red light, the protein acts as a voltage sensor, responding to changes in membrane voltages by producing a flash of light in the presence of an action potential. Although this property could in principle allow Arch to detect the activity of networks of neurons, the light signal marking this neuronal activity was often too dim to see.

To fix this problem, Arnold and her colleagues made the Arch protein brighter using a method called directed evolution—a technique Arnold originally pioneered in the early 1990s. The researchers introduced mutations into the Arch gene, thus encoding millions of variants of the protein. They transferred the mutated genes into E. coli cells, which produced the mutant proteins encoded by the genes. They then screened thousands of the resulting E. coli colonies for the intensities of their fluorescence. The genes for the brightest versions were isolated and subjected to further rounds of mutagenesis and screening until the bacteria produced proteins that were 20 times brighter than the original Arch protein.

A paper describing the process and the bright new protein variants that were created was published in the September 9 issue of the Proceedings of the National Academy of Science.

"This experiment demonstrates how rapidly these remarkable bacterial proteins can evolve in response to new demands. But even more exciting is what they can do in neurons, as Viviana discovered," says Arnold.

In a separate study led by Gradinaru's graduate students Nicholas Flytzanis and Claire Bedbrook, who is also advised by Arnold, the researchers genetically incorporated the new, brighter Arch variants into rodent neurons in culture to see which of these versions was most sensitive to voltage changes—and therefore would be the best at detecting action potentials. One variant, Archer1, was not only bright and sensitive enough to mark action potentials in mammalian neurons in real time, it could also be used to identify which neurons were synaptically connected—and communicating with one another—in a circuit.

The work is described in a study published on September 15 in the journal Nature Communications.

"What was interesting is that we would see two cells over here light up, but not this one over there—because the first two are synaptically connected," Gradinaru says. "This tool gave us a way to observe a network where the perturbation of one cell affects another."

However, sensing activity in a living organism and correlating this activity with behavior remained the biggest challenge. To accomplish this goal Gradinaru's team worked with Paul Sternberg, the Thomas Hunt Morgan Professor of Biology, to test Archer1 as a sensor in a living organism—the tiny nematode worm C. elegans. "There are a few reasons why we used the worms here: they are powerful organisms for quick genetic engineering and their tissues are nearly transparent, making it easy to see the fluorescent protein in a living animal," she says.

After incorporating Archer1 into neurons that were a part of the worm's olfactory system—a primary source of sensory information for C. elegans—the researchers exposed the worm to an odorant. When the odorant was present, a baseline fluorescent signal was seen, and when the odorant was removed, the researchers could see the circuit of neurons light up, meaning that these particular neurons are repressed in the presence of the stimulus and active in the absence of the stimulus. The experiment was the first time that an Arch variant had been used to observe an active circuit in a living organism.

Gradinaru next hopes to use tools like Archer1 to better understand the complex neuronal networks of mammals, using microbial opsins as sensing and actuating tools in optogenetically modified rodents.

"For the future work it's useful that this tool is bifunctional. Although Archer1 acts as a voltage sensor under red light, with green light, it's an inhibitor," she says. "And so now a long-term goal for our optogenetics experiments is to combine the tools with behavior-controlling properties and the tools with voltage-sensing properties. This would allow us to obtain all-optical access to neuronal circuits. But I think there is still a lot of work ahead."

One goal for the future, Gradinaru says, is to make Archer1 even brighter. Although the protein's fluorescence can be seen through the nearly transparent tissues of the nematode worm, opaque organs such as the mammalian brain are still a challenge. More work, she says, will need to be done before Archer1 could be used to detect voltage changes in the neurons of living, behaving mammals.

And that will require further collaborations with protein engineers and biochemists like Arnold.

"As neuroscientists we often encounter experimental barriers, which open the potential for new methods. We then collaborate to generate tools through chemistry or instrumentation, then we validate them and suggest optimizations, and it just keeps going," she says. "There are a few things that we'd like to be better, and through these many iterations and hard work it can happen."

The work published in both papers was supported with grants from the National Institutes of Health (NIH), including an NIH/National Institute of Neurological Disorders and Stroke New Innovator Award to Gradinaru; Beckman Institute funding for the BIONIC center; grants from the U.S. Army Research Office as well as a Caltech Biology Division Training Grant and startup funds from Caltech's President and Provost, and the Division of Biology and Biological Engineering; and other financial support from the Shurl and Kay Curci Foundation and the Life Sciences Research Foundation.

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Emotions in the Brain: An Interview with David Anderson

This year has been a busy one for biologist David Anderson, Caltech's Seymour Benzer Professor of Biology. In 2014 alone, Anderson's lab has reported finding neurons in the male fly brain that promote fighting and, in the mouse brain, identified a "seesaw" circuit that controls the transition between social and asocial behaviors, neurons that control aggressive behavior, a neural circuit that controls anxiety, and a network of cells that switches appetite on and off.

The flurry of discoveries, made possible using state-of-the-art neurobiology techniques such as optogenetics (a technique that uses light to control neural activity), is the result of years of research by the lab to understand emotions and how they are encoded in the brain. We recently spoke to Anderson about this work, his goals, and how the interdisciplinary collaborations he is building at Caltech are helping to spur a revolution in neuroscience.

 

How would you define an "emotion"?

There has been ongoing debate for decades about what "emotion" means, and there is no generally accepted definition. In an article that Ralph Adolphs [Bren Professor of Psychology and Neuroscience and Professor of Biology] and I recently wrote, we put forth the view that emotions are a type of internal brain state with certain general properties that can exist independently of subjective, conscious experience. That means we can study such brain states in animal models like flies or mice without worrying about whether they are consciously aware or not. We use the behaviors that express those states as a readout. For example, behaviors that express the emotion state we call "fear" are freezing and flight. Behaviors that express "anger" include various forms of aggression.

 

So you study these behaviors to get at the underlying emotion and its neural circuitry?

Ultimately, yes. We use genetically based techniques that have been developed over the last 10 years or so—including but not limited to optogenetics, imaging of brain activity, and mapping of neuronal connections—to try to identify specific populations of neurons in the brain that control these "emotional" behaviors. Are there specific populations of neurons in the brain that control aggression, for example? If so, where are those neurons located in the brain? How do they function? Do they only control behaviors, or do they encode internal states as well?

 

Do you know any of these answers yet?

We have identified, in fruit flies and in mice, small populations of neurons that control aggression. In flies, we have identified a population of as few as three to five neurons that, when activated, are sufficient to make a fly fight.

In the mouse, we have identified an analogous population in a deep brain structure called the hypothalamus. There are about 2,000 of those neurons. Activating these neurons is sufficient to promote aggression, and inhibiting these neurons can stop a fight dead in it tracks.

 

Do you think similar populations of "aggression" neurons are found in humans? Could they be related to problems with violence in people?

We're studying these problems because they are fundamental to understanding how the brain works, but certainly it doesn't escape our attention that violence is a pervasive public health problem. My feeling is that we need to understand the basic brain circuitry that controls aggression if we are ever going to understand abnormal forms of aggression, such as sexual violence.

In that respect, it's interesting that we have discovered, in both flies and mice, small populations of neurons that control both aggression and mating (reproductive) behavior. So in a male mouse, for example, if you optogenetically stimulate these neurons at a lower light intensity, the animal will try to mate instead of fight. At a higher stimulation intensity, the animal switches from mounting to attack. It's amazing to watch.

A really important objective over the next several years is to try to figure out how the brain can keep sex and violence separated if the neurons are so intimately related to each other, starting with the question of whether they are the same or different neurons. Obviously that could have implications for sexual violence, for example. It could be that there are people who, as it were, have their wires crossed in these regions of the brain, and that causes them to express violent behavior inappropriately.

 

With regard to your recent study that identified neurons that function as a "brake" on appetite, could that same kind of mis-wiring contribute to eating disorders?

It could. I think the field as a whole—meaning the field of psychiatry—is moving away from the popular idea that psychiatric disorders are due to chemical imbalances in the brain, as if the brain were a bag of soup flavored with dopamine and serotonin, to the idea that psychiatric disorders are due to dysfunctions of brain circuitry as well as chemistry.

 

You've found a "seesaw circuit" in the amygdala that tips between social behavior and self-directed behavior depending on which of two populations of neurons is active. Did you expect the brain to be wired this way?

No. It was also completely unexpected that these two populations segregate according to the most basic distinction between neurons in the brain: inhibitory neurons and excitatory neurons. Inhibitory neurons control the social behaviors. Excitatory neurons control the self-grooming behaviors. It did not have to be that way.

 

Could the proportion of these neurons explain something like personality—whether a person is introverted or extroverted?

That is a fascinating question—whether differences in the behavior of individuals might reflect differences in the relative numbers of different types of neurons. We're trying to see if that is true in different strains of laboratory mice that show different levels of aggression. It is a new direction of research in my lab.

 

Does the discovery of these kinds of circuits suggest possible treatments for human disorders? Could you alter a circuit to change behavior?

It might be possible that, if you found the right population of neurons, you could override the effect of a gene mutation to promote autism or some other psychiatric disorder by pushing the activity of the circuits in a different direction.

 

Tip the balance of the seesaw . . .

Tip the balance of the seesaw in the other direction.

However, this is very far in the future.

But to take a step back to the 35,000-foot level: All of this is happening in the context of a field-wide revolution in neuroscience, a revolution in technology for understanding the brain at the neural circuit level. When I was on the advisory committee for the Obama BRAIN project, we decided that it should focus on supporting the development of this kind of technology.

The technology—in optics and nanotechnology and molecular biology and genetics—allows us to identify populations of neurons that control behaviors, map their connections, measure their activity during behaviors, and manipulate their function, turning them on and off, with a laser-like precision that we could never do before.

If you think of specific populations as a needle in a haystack, these technologies allow us to see and touch and manipulate the needle separately from the haystack. That doesn't mean it won't affect the haystack, but at least we know what we're doing.

 

Your lab's focus changed as a result of the advent of these new methods. Can you tell us about that?

Around the early 2000s, I decided that this area of neuroscience was going to be ripe for new discoveries, although much of this new technology didn't exist then. Caltech helped me to completely retool my laboratory, to move from the study of brain development and stem cell biology to the study of neural circuits and behavior—a major transition from both the intellectual and technical standpoint. It was sort of like turning a sailboat into a motorboat without stopping moving.

 

Do you have a vision of how the field will develop in the future?

This work is increasingly interdisciplinary. It needs molecular biology. It needs optical physics. It needs nanotechnology. It needs modeling, theory, computer science, and electrical engineering. No one laboratory can be competent in all of these different areas.

What has kept me here at Caltech is the ability to collaborate with people from different disciplinary backgrounds. What I am excited about, going forward, is to try to develop a new style of research here in which several laboratories devote their collective energies toward solving a challenging problem in a collaborative way, that they couldn't do if they just stayed in their silos and did their own thing.

 

Have you already set up some of these kinds of collaborations?

Yes. For example, I've been working since 2009 with Pietro Perona [Allen E. Puckett Professor of Electrical Engineering], who has applied his skills in machine vision and machine learning to figure out how to automatically measure aggressive behaviors in flies. We are trying to develop similar technology for the mouse as well. It is not only enormously labor-saving but opens a new, more quantitative approach to describing behavior. And there is also my collaboration on emotion theory with Ralph Adolphs in the Division of the Humanities and Social Sciences.

One of the strong recommendations of the BRAIN committee was to promote these kinds of interdisciplinary, cross-laboratory projects. I think it is important for Caltech to recognize that because of its strength in computer science, applied physics and engineering, and its strength in neuroscience, psychology and social sciences, it is ideally poised to promote and facilitate collaborations between physical scientists and neuroscientists.

 

Interdisciplinary work is something that Caltech does very well.

It is. But in this area of interdisciplinary neuroscience, we have particularly exciting opportunities to engage faculty in multiple divisions across campus. I think this is an ideal moment for us to seize the opportunities identified by the BRAIN initiative, and take advantage of what we do best.

 

When you say "what we do best," what do you mean?

Nimble, interdisciplinary and creative collaborations between labs, which would be harder to implement at larger institutions. Caltech is perfectly positioned to exploit the revolution in neuroscience, in its own unique and interdisciplinary way—exploiting our growing strength in neuroscience and our traditional strengths in genetics, the physical sciences, and engineering—to solve the enormous challenge of how the brain works.

 

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Slimy Fish and the Origins of Brain Development

Lamprey—slimy, eel-like parasitic fish with tooth-riddled, jawless sucking mouths—are rather disgusting to look at, but thanks to their important position on the vertebrate family tree, they can offer important insights about the evolutionary history of our own brain development, a recent study suggests.

The work appears in a paper in the September 14 advance online issue of the journal Nature.

"Lamprey are one of the most primitive vertebrates alive on Earth today, and by closely studying their genes and developmental characteristics, researchers can learn more about the evolutionary origins of modern vertebrates—like jawed fishes, frogs, and even humans," says paper coauthor Marianne Bronner, the Albert Billings Ruddock Professor of Biology and director of Caltech's unique Zebrafish/Xenopus/Lamprey facility, where the study was done.

The facility is one of the few places in the world where lampreys can be studied in captivity. Although the parasitic lamprey are an invasive pest in the Great Lakes, they are difficult to study under controlled conditions; their lifecycle takes up to 10 years and they only spawn for a few short weeks in the summer before they die.

Each summer, Bronner and her colleagues receive shipments of wild lamprey from Michigan just before the prime of breeding season. When the lamprey arrive, they are placed in tanks where the temperature of the water is adjusted to extend the breeding season from around three weeks to up to two months. In those extra weeks, the lamprey produce tens of thousands of additional eggs and sperm, which, via in vitro fertilization, generate tens of thousands of additional embryos for study. During this time, scientists from all over the world come to Caltech to perform experiments with the developing lamprey embryos.

In the current study, Bronner and her collaborators—who traveled to Caltech from Stower's Institute for Medical Research in Kansas City, Missouri—studied the origins of the vertebrate hindbrain.

The hindbrain is a part of the central nervous system common to chordates—or organisms that have a nerve cord like our spinal cord. During the development of vertebrates—a subtype of chordates that have backbones—the hindbrain is compartmentalized into eight segments, each of which becomes uniquely patterned to establish networks of neuronal circuits. These segments eventually give rise to adult brain regions like the cerebellum, which is important for motor control, and the medulla oblongata, which is necessary for breathing and other involuntary functions.

However, this segmentation is not present in so-called "invertebrate chordates"—a grouping of chordates that lack a backbone, such as sea squirts and lancelets.

"The interesting thing about lampreys is that they occupy an intermediate evolutionary position between the invertebrate chordates and the jawed vertebrates," says Hugo Parker, a postdoc at Stower's Institute and first author on the study. "By investigating aspects of lamprey embryology, we can get a picture of how vertebrate traits might have evolved."

In the vertebrates, segmental patterning genes called Hox genes help to determine the animal's head-to-tail body plan—and those same Hox genes also control the segmentation of the hindbrain. Although invertebrate chordates also have Hox genes, these animals don't have segmented hindbrains. Because lampreys are centered between these two types of organisms on the evolutionary tree, the researchers wanted to know whether or not Hox genes are involved in patterning of the lamprey hindbrain.

To their surprise, the researchers discovered that the lamprey hindbrain was not only segmented during development but the process also involved Hox genes—just like in its jawed vertebrate cousins.

"When we started, we thought that the situation was different, and the Hox genes were not really integrated into the process of segmentation as they are in jawed vertebrates," Parker says. "But in actually doing this project, we discovered the way that lamprey Hox genes are expressed and regulated is very similar to what we see in jawed vertebrates." This means that hindbrain segmentation—and the role of Hox genes in this segmentation—happened earlier on in evolution than was once thought, he says.

Parker, who has been spending his summers at Caltech studying lampreys since 2008, is next hoping to pinpoint other aspects of the lamprey hindbrain that may be conserved in modern vertebrates—information that will help contribute to a fundamental understanding of vertebrate development. And although those investigations will probably mean following the lamprey for a few more summers at Caltech, Parker says his time in the lamprey facility continually offers a one-of-a-kind experience.

"The lamprey system here is unique in the world—and it's not just the water tanks and how we've learned to maintain the animals. It's the small nucleus of people who have particular skills, people who come in from all over the world to work together, share protocols, and develop the field together," he says. "That's one of the things I've liked ever since I first came here. I really felt like I was a part of something very special.

These results were published in a paper titled "A Hox regulatory network of hindbrain segmentation is conserved to the base of vertebrates." Robb Krumlauf, a scientific director at the Stower's Institute and professor at the Kansas University Medical Center, was also a coauthor on the study. The Zebrafish/Xenopus/Lamprey facility at Caltech is a Beckman Institute facility.

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