Mapping the cellular circuits behind spitting

For over a decade, researchers have known that the roundworm Caenorhabditis elegans can detect and avoid short-wavelength light, despite lacking eyes and the light-absorbing molecules required for sight. As a graduate student in the Horvitz lab, Nikhil Bhatla proposed an explanation for this ability. He observed that light exposure not only made the worms wriggle away, but it also prompted them to stop eating. This clue led him to a series of studies that suggested that his squirming subjects weren’t seeing the light at all — they were detecting the noxious chemicals it produced, such as hydrogen peroxide. Soon after, the Horvitz lab realized that worms not only taste the nasty chemicals light generates, they also spit them out.

Now, in a study recently published in eLife, a team led by former graduate student Steve Sando reports the mechanism that underlies spitting in C. elegans. Individual muscle cells are generally regarded as the smallest units that neurons can independently control, but the researchers’ findings question this assumption. In the case of spitting, they determined that neurons can direct specialized subregions of a single muscle cell to generate multiple motions — expanding our understanding of how neurons control muscle cells to shape behavior.

“Steve made the remarkable discovery that the contraction of a small region of a particular muscle cell can be uncoupled from the contraction of the rest of the same cell,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, Howard Hughes Medical Institute Investigator, and senior author of the study. “Furthermore, Steve found that such subcellular muscle compartments can be controlled by neurons to dramatically alter behavior.”

Roundworms are like vacuum cleaners that wiggle around hoovering up bacteria. The worm’s mouth, also known as the pharynx, is a muscular tube that traps the food, chews it, and then transfers it to the intestines through a series of “pumping” contractions.

Researchers have known for over a decade that worms flee from UV, violet, or blue light. But Bhatla discovered that this light also interrupts the constant pumping of the pharynx, because the taste produced by the light is so nasty that the worms pause feeding. As he looked closer, Bhatla noticed the worms’ response was actually quite nuanced. After an initial pause, the pharynx briefly starts pumping again in short bursts before fully stopping — almost like the worm was chewing for a bit even after tasting the unsavory light. Sometimes, a bubble would escape from the mouth, like a burp.

After he joined the project, Sando discovered that the worms were neither burping nor continuing to munch. Instead, the “burst pumps” were driving material in the opposite direction, out of the mouth into the local environment, rather than further back into the pharynx and intestine. In other words, the bad-tasting light caused worms to spit. Sando then spent years chasing his subjects around the microscope with a bright light and recording their actions in slow motion, in order to pinpoint the neural circuitry and muscle motions required for this behavior.

“The discovery that the worms were spitting was quite surprising to us, because the mouth seemed to be moving just like it does when it’s chewing,” Sando says. “It turns out that you really needed to zoom in and slow things down to see what’s going on, because the animals are so small and the behavior is happening so quickly.”

To analyze what’s happening in the pharynx to produce this spitting motion, the researchers used a tiny laser beam to surgically remove individual nerve and muscle cells from the mouth and discern how that affected the worm’s behavior. They also monitored the activity of the cells in the mouth by tagging them with specially-engineered fluorescent “reporter” proteins.

They saw that while the worm is eating, three muscle cells towards the front of the pharynx called pm3s contract and relax together in synchronous pulses. But as soon as the worm tastes light, the subregions of these individual cells closest to the front of the mouth become locked in a state of contraction, opening the front of the mouth and allowing material to be propelled out. This reverses the direction of the flow of the ingested material and converts feeding into spitting.

The team determined that this “uncoupling” phenomenon is controlled by a single neuron at the back of the worm’s mouth. Called M1, this nerve cell spurs a localized influx of calcium at the front end of the pm3 muscle likely responsible for triggering the sub-cellular contractions.

M1 relays important information like a switchboard. It receives incoming signals from many different neurons, and transmits that information to the muscles involved in spitting. Sando and his team suspect that the strength of the incoming signal can tune the worm’s behavior in response to tasting light. For instance, their findings suggest that a revolting taste elicits a vigorous rinsing of the mouth, while a mildly unpleasant sensation causes the worm spit more gently, just enough to eject the contents.

In the future, Sando thinks the worm could be used as a model to study how neurons trigger subregions of muscle cells to constrict and shape behavior — a phenomenon they suspect occurs in other animals, possibly including humans.

“We’ve essentially found a new way for a neuron to move a muscle,” Sando says. “Neurons orchestrate the motions of muscles, and this could be a new tool that allows them to exert a sophisticated kind of control. That’s pretty exciting.”

Biologists discover a trigger for cell extrusion

For all animals, eliminating some cells is a necessary part of embryonic development. Living cells are also naturally sloughed off in mature tissues; for example, the lining of the intestine turns over every few days.

One way that organisms get rid of unneeded cells is through a process called extrusion, which allows cells to be squeezed out of a layer of tissue without disrupting the layer of cells left behind. MIT biologists have now discovered that this process is triggered when cells are unable to replicate their DNA during cell division.

The researchers discovered this mechanism in the worm C. elegans, and they showed that the same process can be driven by mammalian cells; they believe extrusion may serve as a way for the body to eliminate cancerous or precancerous cells.

“Cell extrusion is a mechanism of cell elimination used by organisms as diverse as sponges, insects, and humans,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, a Howard Hughes Medical Institute investigator, and the senior author of the study. “The discovery that extrusion is driven by a failure in DNA replication was unexpected and offers a new way to think about and possibly intervene in certain diseases, particularly cancer.”

MIT postdoc Vivek Dwivedi is the lead author of the paper, which appears today in Nature. Other authors of the paper are King’s College London research fellow Carlos Pardo-Pastor, MIT research specialist Rita Droste, MIT postdoc Ji Na Kong, MIT graduate student Nolan Tucker, Novartis scientist and former MIT postdoc Daniel Denning, and King’s College London professor of biology Jody Rosenblatt.

Stuck in the cell cycle

In the 1980s, Horvitz was one of the first scientists to analyze a type of programmed cell suicide called apoptosis, which organisms use to eliminate cells that are no longer needed. He made his discoveries using C. elegans, a tiny nematode that contains exactly 959 cells. The developmental lineage of each cell is known, and embryonic development follows the same pattern every time. Throughout this developmental process, 1,090 cells are generated, and 131 cells undergo programmed cell suicide by apoptosis.

Horvitz’s lab later showed that if the worms were genetically mutated so that they could not eliminate cells by apoptosis, a few of those 131 cells would instead be eliminated by cell extrusion, which appears to be able to serve as a backup mechanism to apoptosis. How this extrusion process gets triggered, however, remained a mystery.

To unravel this mystery, Dwivedi performed a large-scale screen of more than 11,000 C. elegans genes. One by one, he and his colleagues knocked down the expression of each gene in worms that could not perform apoptosis. This screen allowed them to identify genes that are critical for turning on cell extrusion during development.

To the researchers’ surprise, many of the genes that turned up as necessary for extrusion were involved in the cell division cycle. These genes were primarily active during first steps of the cell cycle, which involve initiating the cell division cycle and copying the cell’s DNA.

Further experiments revealed that cells that are eventually extruded do initially enter the cell cycle and begin to replicate their DNA. However, they appear to get stuck in this phase, leading them to be extruded.

Most of the cells that end up getting extruded are unusually small, and are produced from an unequal cell division that results in one large daughter cell and one much smaller one. The researchers showed that if they interfered with the genes that control this process, so that the two daughter cells were closer to the same size, the cells that normally would have been extruded were able to successfully complete the cell cycle and were not extruded.

The researchers also showed that the failure of the very small cells to complete the cell cycle stems from a shortage of the proteins and DNA building blocks needed to copy DNA. Among other key proteins, the cells likely don’t have enough of an enzyme called LRR-1, which is critical for DNA replication. When DNA replication stalls, proteins that are responsible for detecting replication stress quickly halt cell division by inactivating a protein called CDK1. CDK1 also controls cell adhesion, so the researchers hypothesize that when CDK1 is turned off, cells lose their stickiness and detach, leading to extrusion.

Cancer protection

Horvitz’s lab then teamed up with researchers at King’s College London, led by Rosenblatt, to investigate whether the same mechanism might be used by mammalian cells. In mammals, cell extrusion plays an important role in replacing the lining of the intestines, lungs, and other organs.

The researchers used a chemical called hydroxyurea to induce DNA replication stress in canine kidney cells grown in cell culture. The treatment quadrupled the rate of extrusion, and the researchers found that the extruded cells made it into the phase of the cell cycle where DNA is replicated before being extruded. They also showed that in mammalian cells, the well-known cancer suppressor p53 is involved in initiating extrusion of cells experiencing replication stress.

That suggests that in addition to its other cancer-protective roles, p53 may help to eliminate cancerous or precancerous cells by forcing them to extrude, Dwivedi says.

“Replication stress is one of the characteristic features of cells that are precancerous or cancerous. And what this finding suggests is that the extrusion of cells that are experiencing replication stress is potentially a tumor suppressor mechanism,” he says.

The fact that cell extrusion is seen in so many animals, from sponges to mammals, led the researchers to hypothesize that it may have evolved as a very early form of cell elimination that was later supplanted by programmed cell suicide involving apoptosis.

“This cell elimination mechanism depends only on the cell cycle,” Dwivedi says. “It doesn’t require any specialized machinery like that needed for apoptosis to eliminate these cells, so what we’ve proposed is that this could be a primordial form of cell elimination. This means it may have been one of the first ways of cell elimination to come into existence, because it depends on the same process that an organism uses to generate many more cells.”

Dwivedi, who earned his PhD at MIT, was a Khorana scholar before entering MIT for graduate school. This research was supported by the Howard Hughes Medical Institute and the National Institutes of Health.

Eyeless roundworms sense color

Roundworms don’t have eyes or the light-absorbing molecules required to see. Yet, new research shows they can somehow sense color. The study, published on March 5 in the journal Science, suggests worms use this ability to assess the risk of feasting on potentially dangerous bacteria that secrete blue toxins. The researchers pinpointed two genes that contribute to this spectral sensitivity and are conserved across many organisms, including humans.

“It’s amazing to me that a tiny worm — with neither eyes nor the molecular machinery used by eyes to detect colors — can identify and avoid a toxic bacterium based, in part, on its blue color,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, Howard Hughes Medical Institute Investigator, and the co-senior author of the study.

“One of the joys of being a biologist is the opportunity to discover things about nature that no one has ever imagined before,” says Horvitz.

A model organism

The roundworm in question, Caenorhabditis elegans, is only about a millimeter long. Despite their minute stature and simple nervous system, these nematodes display a complex repertoire of behaviors. They can smell, taste, sense touch, react to temperature, and even escape or change their feeding patterns in response to bright, blue light. Although researchers once thought that these worms bury themselves deep in soil, it’s becoming increasingly clear that C. elegans prefers compost heaps above ground that offer some sun exposure. As a result, roundworms may have a need for light- and color-sensing capabilities after all.

The decomposing organic matter where C. elegans resides offers an array of scrumptious microbes, including bacteria like Pseudomonas aeruginosa, which secretes a distinctive blue toxin. Previous studies showed that worms in the lab feed on a lawn of P. aeruginosa for a few hours and then begin avoiding their food — perhaps because the bacteria continue to divide and excrete more of the colorful poison. Dipon Ghosh, Horvitz lab postdoc and the study’s first author, wondered whether the worms were using the distinctive color to determine if their meal was too toxic to consume.

A spectrum of behavior

Over the course of his experiments, Ghosh noticed that his worms were more likely to flee the colorful bacterial lawn if it was bathed in white light from a nearby LED bulb. This finding was curious on its own, but Ghosh wanted know if the blue toxin played a role as well.

To test this theory, he first exchanged the blue toxin for a harmless dye of the same color, and then for a clear, colorless toxin. On its own, neither substitute was sufficient to spur avoidance. Only together did they prompt a response — suggesting the worms were assessing both the toxic nature and the color of the P. aeruginosa secretions simultaneously. Once again, this behavioral pattern only emerged in the presence of the LED’s white light.

To test how worms sense color, the researchers placed C. elegans on an agar plate under tinted lights. Image: Eugene Lee

Intrigued, Ghosh wanted to examine what it was about the blue color that triggered avoidance. This time, he used two colored LED lights, one blue and one amber, to tint the ambient light. In doing so, he could control the ratio of wavelengths without changing the total energy delivered to the worms. The beam had previously contained the entire visible spectrum, but mixing the amber and blue bulbs allowed Ghosh to tweak the relative amounts of short-wavelength blue light and long-wavelength amber light. Surprisingly, the worms only fled the bacterial lawn when their environment was bathed in light with specific blue:amber ratios.

“We were able to definitively show that worms aren’t sensing the world in grayscale and simply evaluating the levels of brightness and darkness,” Ghosh says. “They’re actually comparing ratios of wavelengths and using that information to make decisions — which was thoroughly unexpected.”

It wasn’t until Ghosh ran his experiments again, this time using various types of wild C. elegans, that he realized the popular laboratory strain he’d been using was actually less color-sensitive compared to its close relatives. After analyzing the genomes of these worms, he was able to identify two genes in particular (called jkk-1 and lec-3) that contributed to these variations in color-dependent foraging.

Although the two genes play many important functions in a variety of organisms, including humans, they are both involved in molecular pathways that help cells respond to stress caused by damaging ultraviolet light.

“We’ve discovered that the color of light in the worm’s environment can influence how the worm navigates the world,” Ghosh says. “But our work suggests that many genes, in addition to the two we’ve already identified, can affect color sensitivity, and we’re now exploring how.”

Nature’s innovation

The notion that worms can sense color is “astounding” and showcases nature’s innovation, according to Leslie Vosshall, Robin Chemers Neustein Professor and Howard Hughes Medical Institute Investigator at The Rockefeller University, who was not involved in the study. “These worms are sliding around in a dim muck with colorful, toxic bacteria. It would be helpful to see and avoid them, so the worms somehow evolved a completely new way to see.”

Vosshall is curious about which cells in C. elegans help discriminate light, as well as the specific roles that the jkk-1 and lec-3 genes play in mediating light perception. “This paper, like all important papers, raises many additional questions,” she says.

Ghosh suspects the lab’s findings could generalize to other critters besides roundworms. If nothing else, it’s clear that light-sensitivity does not always require vision — or eyes. C. elegans are seeing the light, and now so are the biologists.

This research was funded by the Howard Hughes Medical Institute and National Institute of General Medical Sciences.

Storytelling brings MIT neuroscience community together

When the coronavirus pandemic shut down offices, labs, and classrooms across the MIT campus last spring, many members of the MIT community found it challenging to remain connected to one another in meaningful ways. Motivated by a desire to bring the neuroscience community back together, the McGovern Institute hosted a virtual storytelling competition featuring a selection of postdocs, grad students, and staff from across the institute.

“This has been an unprecedented year for us all,” says McGovern Institute Director Robert Desimone. “It has been twenty years since Pat and Lore McGovern founded the McGovern Institute, and despite the challenges this anniversary year has brought to our community, I have been inspired by the strength and perseverance demonstrated by our faculty, postdocs, students and staff. The resilience of this neuroscience community – and MIT as a whole – is indeed something to celebrate.”

The McGovern Institute had initially planned to hold a large 20th anniversary celebration in the atrium of Building 46 in the fall of 2020, but the pandemic made a gathering of this size impossible. The institute instead held a series of virtual events, including the November 12 story slam on the theme of resilience.

H. Robert Horvitz

Learning from Worms

Bob Horvitz studies the nematode worm Caenorhabditis elegans. Only 1 mm long and containing fewer than 1000 cells, C. elegans has been key to discovering fundamental biological mechanisms that are conserved across species. Horvitz has focused on the genetic control of animal development and behavior, and on the mechanisms that underlie neurodegenerative disease. By identifying mutations that affect C. elegans behavior, Horvitz has revealed much about the genetic control of many aspects of nervous system development and of brain function, including how neural circuits control specific behaviors and how behavior is modulated by experience and by the environment.


Biologists discover function of gene linked to familial ALS

MIT biologists have discovered a function of a gene that is believed to account for up to 40 percent of all familial cases of amyotrophic lateral sclerosis (ALS). Studies of ALS patients have shown that an abnormally expanded region of DNA in a specific region of this gene can cause the disease.

In a study of the microscopic worm Caenorhabditis elegans, the researchers found that the gene has a key role in helping cells to remove waste products via structures known as lysosomes. When the gene is mutated, these unwanted substances build up inside cells. The researchers believe that if this also happens in neurons of human ALS patients, it could account for some of those patients’ symptoms.

“Our studies indicate what happens when the activities of such a gene are inhibited — defects in lysosomal function. Certain features of ALS are consistent with their being caused by defects in lysosomal function, such as inflammation,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, and the senior author of the study.

Mutations in this gene, known as C9orf72, have also been linked to another neurodegenerative brain disorder known as frontotemporal dementia (FTD), which is estimated to affect about 60,000 people in the United States.

“ALS and FTD are now thought to be aspects of the same disease, with different presentations. There are genes that when mutated cause only ALS, and others that cause only FTD, but there are a number of other genes in which mutations can cause either ALS or FTD or a mixture of the two,” says Anna Corrionero, an MIT postdoc and the lead author of the paper, which appears in the May 3 issue of the journal Current Biology.

Genetic link

Scientists have identified dozens of genes linked to familial ALS, which occurs when two or more family members suffer from the disease. Doctors believe that genetics may also be a factor in nonfamilial cases of the disease, which are much more common, accounting for 90 percent of cases.

Of all ALS-linked mutations identified so far, the C9orf72 mutation is the most prevalent, and it is also found in about 25 percent of frontotemporal dementia patients. The MIT team set out to study the gene’s function in C. elegans, which has an equivalent gene known as alfa-1.

In studies of worms that lack alfa-1, the researchers discovered that defects became apparent early in embryonic development. C. elegans embryos have a yolk that helps to sustain them before they hatch, and in embryos missing alfa-1, the researchers found “blobs” of yolk floating in the fluid surrounding the embryos.

This led the researchers to discover that the gene mutation was affecting the lysosomal degradation of yolk once it is absorbed into the cells. Lysosomes, which also remove cellular waste products, are cell structures which carry enzymes that can break down many kinds of molecules.

When lysosomes degrade their contents — such as yolk — they are reformed into tubular structures that split, after which they are able to degrade other materials. The MIT team found that in cells with the alfa-1 mutation and impaired lysosomal degradation, lysosomes were unable to reform and could not be used again, disrupting the cell’s waste removal process.

“It seems that lysosomes do not reform as they should, and material accumulates in the cells,” Corrionero says.

For C. elegans embryos, that meant that they could not properly absorb the nutrients found in yolk, which made it harder for them to survive under starvation conditions. The embryos that did survive appeared to be normal, the researchers say.

Robert Brown, chair of the Department of Neurology at the University of Massachusetts Medical School, describes the study as a major contribution to scientists’ understanding of the normal function of the C9orf72 gene.

“They used the power of worm genetics to dissect very fully the stages of vesicle maturation at which this gene seems to play a major role,” says Brown, who was not involved in the study.

Neuronal effects

The researchers were able to partially reverse the effects of alfa-1 loss in the C. elegans embryos by expressing the human protein encoded by the C9orf72 gene. “This suggests that the worm and human proteins are performing the same molecular function,” Corrionero says.

If loss of C9orf72 affects lysosome function in human neurons, it could lead to a slow, gradual buildup of waste products in those cells. ALS usually affects cells of the motor cortex, which controls movement, and motor neurons in the spinal cord, while frontotemporal dementia affects the frontal areas of the brain’s cortex.

“If you cannot degrade things properly in cells that live for very long periods of time, like neurons, that might well affect the survival of the cells and lead to disease,” Corrionero says.

Many pharmaceutical companies are now researching drugs that would block the expression of the mutant C9orf72. The new study suggests certain possible side effects to watch for in studies of such drugs.

“If you generate drugs that decrease C9orf72 expression, you might cause problems in lysosomal homeostasis,” Corrionero says. “In developing any drug, you have to be careful to watch for possible side effects. Our observations suggest some things to look for in studying drugs that inhibit C9orf72 in ALS/FTD patients.”

The research was funded by an EMBO postdoctoral fellowship, an ALS Therapy Alliance grant, a gift from Rose and Douglas Barnard ’79 to the McGovern Institute, and a gift from the Halis Family Foundation to the MIT Aging Brain Initiative.

From cancer to brain research: learning from worms

In Bob Horvitz’s lab, students watch tiny worms as they wriggle under the microscope. Their tracks twist and turn in every direction, and to a casual observer the movements appear random. There is a pattern, however, and the animals’ movements change depending on their environment and recent experiences.

“A hungry worm is different from a well-fed worm,” says Horvitz, David H. Koch Professor of Biology and a McGovern Investigator. “If you consider worm psychology, it seems that the thing in life worms care most about is food.”

Horvitz’s work with the nematode worm Caenorhabditis elegans extends back to the mid-1970s. He was among the first to recognize the value of this microscopic organism as a model species for asking fundamental questions about biology and human disease.

The leap from worm to human might seem great and perilous, but in fact they share many fundamental biological mechanisms, one of which is programmed cell death, also known as apoptosis. Horvitz shared the Nobel Prize in Physiology or Medicine in 2002 for his studies of cell death, which is central to a wide variety of human diseases, including cancer and neurodegenerative disorders. He has continued to study the worm ever since, contributing to many areas of biology but with a particular emphasis on the nervous system and the control of behavior.

In a recently published study, the Horvitz lab has found another fundamental mechanism that likely is shared with mice and humans. The discovery began with an observation by former graduate student Beth Sawin as she watched worms searching for food. When a hungry worm detects a food source, it slows almost to a standstill, allowing it to remain close to the food.
Postdoctoral scientist Nick Paquin analyzed how a mutation in a gene called vps-50, causes worms to slow similarly even when they are well fed. It seemed that these mutant worms were failing to transition normally between the hungry and the well-fed state.

Paquin decided to study the gene further, in worms and also in mouse neurons, the latter in collaboration with Yasunobu Murata, a former research scientist in Martha Constantine-Paton’s lab at the McGovern Institute. The team, later joined by postdoctoral fellow Fernando Bustos in the Constantine-Paton lab, found that the VPS-50 protein controls the activity of synapses, the junctions between nerve cells. VPS-50 is involved in a process that acidifies synaptic vesicles, microscopic bubbles filled with neurotransmitters that are released from nerve terminals, sending signals to other nearby neurons.

If VPS-50 is missing, the vesicles do not mature properly and the signaling from neurons is abnormal. VPS-50 has remained relatively unchanged during evolution, and the mouse version can
substitute for the missing worm gene, indicating the worm and mouse proteins are similar not only in sequence but also in function. This might seem surprising given the wide gap between the tiny nervous system of the worm and the complex brains of mammals. But it is not surprising to Horvitz, who has committed about half of his lab resources to studying the worm’s nervous system and behavior.

“Our finding underscores something that I think is crucially important,” he says. “A lot of biology is conserved among organisms that appear superficially very different, which means that the
understanding and treatment of human diseases can be advanced by studies of simple organisms like worms.”

Human connections

In addition to its significance for normal synaptic function, the vps-50 gene might be important in autism spectrum disorder. Several autism patients have been described with deletions that include vps-50, and other lines of evidence also suggest a link to autism. “We think this is going to be a very important molecule in mammals,” says Constantine-Paton. “We’re now in a position to look into the function of vps-50 more deeply.”

Horvitz and Constantine-Paton are married, and they had chatted about vps-50 long before her lab began to study it. When it became clear that the mutation was affecting worm neurons in a novel way, it was a natural decision to collaborate and study the gene in mice. They are currently working to understand the role of VPS-50 in mammalian brain function, and to explore further the possible link to autism.

The day the worm turned

A latecomer to biology, Horvitz studied mathematics and economics as an undergraduate at MIT in the mid-1960s. During his last year, he took a few biology classes and then went on to earn
a doctoral degree in the field at Harvard University, working in the lab of James Watson (of double helix fame) and Walter Gilbert. In 1974, Horvitz moved to Cambridge, England, where he worked with Sydney Brenner and began his studies of the worm.

“Remarkably, all of my advisors, even my undergraduate advisor in economics here at MIT, Bob Solow, now have Nobel Prizes,” he notes.

The comment is matter-of-fact, and Horvitz is anything but pretentious. He thinks about both big questions and small experimental details and is always on the lookout for links between the
worm and human health.

“When someone in the lab finds something new, Bob is quick to ask if it relates to human disease,” says former graduate student Nikhil Bhatla. “We’re not thinking about that. We’re deep in
the nitty-gritty, but he’s directing us to potential collaborators who might help us make that link.”

This kind of mentoring, says Horvitz, has been his primary role since he joined the MIT faculty in 1978. He has trained many of the current leaders in the worm field, including Gary Ruvkun
and Victor Ambros, who shared the 2008 Lasker Award, Michael Hengartner, now President of the University of Zurich, and Cori Bargmann, who recently won the McGovern’s 2016 Scolnick Prize in Neuroscience.

“If the science we’ve done has been successful, it’s because I’ve been lucky to have outstanding young researchers as colleagues,” Horvitz says.

Before becoming a mentor, Horvitz had to become a scientist himself. At Harvard, he studied bacterial viruses and learned that even the simplest organisms could provide valuable insights about fundamental biological processes.

The move to Brenner’s lab in Cambridge was a natural step. A pioneer in the field of molecular biology, Brenner was also the driving force behind the adoption of C. elegans as a genetic model organism, which he advocated for its simplicity (adults have fewer than 1000 cells, and only 302 neurons) and short generation time (only three days). Working in Brenner’s lab, Horvitz
and his collaborator John Sulston traced the lineage of every body cell from fertilization to adulthood, showing that the sequence of cell divisions was the same in each individual animal. Their landmark study provided a foundation for the entire field. “They know all the cells in the worm. Every single one,” says Constantine-Paton. “So when they make a mutation and something is weird, they can determine precisely which cell or set of cells are affected. We can only dream of having such an understanding of a mammal.”

It is now known that the worm has about 20,000 genes, many of which are conserved in mammals including humans. In fact, in many cases, a cloned human gene can stand in for a missing
worm gene, as is the case for vps-50. As a result, the worm has been a powerful discovery machine for human biology. In the early years, though, many doubted whether worms would be relevant. Horvitz persisted undeterred, and in 1992 his conviction paid off, with the discovery of ced-9, a worm gene that regulates programmed cell death. A graduate student in Horvitz’ lab cloned ced-9 and saw that it resembled a human cancer gene called Bcl-2. They also showed that human Bcl-2 could substitute for a mutant ced-9 gene in the worm and concluded that the two genes have similar functions: ced-9 in worms protects healthy cells from death, and Bcl-2 in cancer patients protects cancerous cells from death, allowing them to multiply. “This was the moment we knew that the studies we’d been doing with C. elegans were going to be relevant to understanding human biology and disease,” says Horvitz.

Ten years later, in 2002, he was in the French Alps with Constantine-Paton and their daughter Alex attending a wedding, when they heard the news on the radio: He’d won a Nobel Prize, along with Brenner and Sulston. On the return trip, Alex, then 9 years old but never shy, asked for first-class upgrades at the airport; the agent compromised and gave them all upgrades to business class instead.

Discovery machine at work

Since the Nobel Prize, Horvitz has studied the nervous system using the same strategy that had been so successful in deciphering the mechanism of programmed cell death. His approach, he says, begins with traditional genetics. Researchers expose worms to mutagens and observe their behavior. When they see an interesting change, they identify the mutation and try to link the gene to the nervous system to understand how it affects behavior.

“We make no assumptions,” he says. “We let the animal tell us the answer.”

While Horvitz continues to demonstrate that basic research using simple organisms produces invaluable insights about human biology and health, there are other forces at work in his lab. Horvitz maintains a sense of wonder about life and is undaunted by big questions.

For instance, when Bhatla came to him wanting to look for evidence of consciousness in worms, Horvitz blinked but didn’t say no. The science Bhatla proposed was novel, and the question
was intriguing. Bhatla pursued it. But, he says, “It didn’t work.”

So Bhatla went back to the drawing board. During his earlier experiments, he had observed that worms would avoid light, a previously known behavior. But he also noticed that they immediately stopped feeding. The animals had provided a clue. Bhatla went on to discover that worms respond to light by producing hydrogen peroxide, which activates a taste receptor.

In a sense, worms taste light, a wonder of biology no one could have predicted.

Some years ago, the Horvitz lab made t-shirts displaying a quote from the philosopher Friedrich Nietzsche: “You have made your way from worm to man, and much within you is still worm.”
The words have become an informal lab motto, “truer than Nietzsche could everhave imagined,” says Horvitz. “There’s still so much mystery, particularly about the brain, and we are still learning from the worm.”

Neuroscientists discover a gene that controls worms’ behavioral state

In a study of worms, MIT neuroscientists have discovered a gene that plays a critical role in controlling the switch between alternative behavioral states, which for humans include hunger and fullness, or sleep and wakefulness.

This gene, which the researchers dubbed vps-50, helps to regulate neuropeptides — tiny proteins that carry messages between neurons or from neurons to other cells. This kind of signaling is important for controlling physiology and behavior in animals, including humans. Deletions of the human counterpart of the vps-50 gene have been found in some people with autism.

“Given what is reported in this paper about how the gene works, coupled with findings by others concerning the genetics of autism, we suggest that the disruption of the function of this gene could promote autism,” says H. Robert Horvitz, the David H. Koch Professor of Biology and a member of MIT’s McGovern Institute for Brain Research.

Horvitz and Martha Constantine-Paton, an MIT professor of brain and cognitive sciences and member of the McGovern Institute, are the senior authors of the study, which appears in the March 3 issue of the journal Current Biology. The paper’s lead authors are former MIT postdocs Nicolas Paquin and Yasunobu Muruta.

Influencing behavior

Neuropeptides, which are involved in brain functions such as reward, metabolism, and learning and memory, are released from cellular structures called dense-core vesicles.

In the new study, the researchers found that the vps-50 gene encodes a protein that is important in the generation of such vesicles and in the release of neuropeptides from them.

They discovered the protein in the worm Caenorhabditis elegans, where it is found primarily in nerve cells. In those cells, vps-50 associates with both synaptic vesicles and dense-core vesicles, which release neurotransmitters such as dopamine and serotonin. The researchers showed that vps-50 is required for maturation of the dense-core vesicles and also regulates activity of a proton pump that acidifies the vesicles. Without the proper acidity level, the vesicles’ ability to produce neuropeptides is impaired.

The researchers also found distinctive behavioral effects in C. elegans worms, which normally change their speed depending on food availability and whether they have recently eaten.

“Worms are the fastest when food (bacteria) is absent, presumably because they are looking for food,” Paquin says. “When they reach food, they slow down, but when you make them hungry for 30 minutes before putting them on food, they slow down even more.”

Worms lacking vps-50 behaved as if they were hungry — moving slowly through a food-rich area even when they were well fed, the researchers found. This suggests that the worms without vps-50 are unable to signal that they are full and continue to behave as if they are hungry. The researchers also found an equivalent gene in mice and showed that it can compensate for loss of the worm version of vps-50, showing that the two genes have the same function.

Human link

One important question raised by the study is how the mouse and human versions of vps-50 affect behavior in those animals, Horvitz says. Although this study focused on switching between hunger and fullness, neuropeptide signaling has been previously shown to control other alternative behaviors such as sleep and wakefulness and also to control social behaviors, such as anxiety.

The researchers suggest that studies of vps-50 might shed light on aspects of autism, because the human version of the gene is missing in some people with autism. Furthermore, a protein known as UNC-31, which is also located in dense-core vesicles has also been linked with autism in humans and mice. When mutated in worms, UNC-31 produces behavioral effects similar to those caused by vps-50 mutations.

“For these reasons, we hope that our studies of vps-50 will provide insights into human neuropsychiatric disorders,” Horvitz says.

The research was funded by the National Institutes of Health and the Simons Center for the Social Brain at MIT.

Tasting light

Human taste receptors are specialized to distinguish several distinct compounds: sugars taste sweet, salts taste salty, and acidic compounds taste sour. Now a new study from MIT finds that the worm Caenorhabditis elegans has taken its powers of detection a step further: The worm can taste hydrogen peroxide, triggering it to stop eating the potentially dangerous substance.

Being able to taste hydrogen peroxide allows the worm to detect light, which generates hydrogen peroxide and other harmful reactive oxygen compounds both within the worm and in its environment.

“This is potentially a brand-new mechanism of sensing light,” says Nikhil Bhatla, the lead author of the paper and a postdoc in MIT’s Department of Biology. “All of the mechanisms of light detection we know about involve a chromophore — a small molecule that absorbs a photon and changes shape or transfers electrons. This seems to be the first example of behavioral light-sensing that requires the generation of a chemical in the process of detecting the light.”

Bhatla and Robert Horvitz, the David H. Koch Professor of Biology, describe the new hydrogen peroxide taste receptors in the Jan. 29 online issue of the journal Neuron.

Though it is not yet known whether there is a human equivalent of this system, the researchers say their discovery lends support to the idea that there may be human taste receptors dedicated to flavors other than the five canonical ones — sweet, salty, bitter, sour, and savory. It also opens the possibility that humans might be able to sense light in ways that are fundamentally different from those known to act in vision.

“I think we have underestimated our biological abilities,” Bhatla says. “Aside from those five, there are other flavors, such as burnt. How do we taste something as burnt? Or what about spicy, or metallic, or smoky? There’s this whole new area that hasn’t really been explored.”

Beyond bitter and sweet

One of the major functions of the sense of taste is to determine whether something is safe, or advantageous, to eat. For humans and other animals, bitterness often serves as a warning of poison, while sweetness can help to identify foods that are rich in energy.

For worms, hydrogen peroxide can be harmful because it can cause extensive cellular trauma, including damaging proteins, DNA, and other molecules in the body. In fact, certain strains of bacteria produce hydrogen peroxide that can kill C. elegans after being eaten. Worms might also ingest hydrogen peroxide from the soil where they live.

Bhatla and Horvitz found that worms stop eating both when they taste hydrogen peroxide and when light shines on them — especially high-energy light, such as violet or ultraviolet. The authors found the exact same feeding response when worms were exposed to either hydrogen peroxide or light, which suggested to them that the same mechanism might be controlling responses to both stimuli.

Worms are known to be averse to light: Previous research by others has shown that they flee when light shines on them. Bhatla and Horvitz have now found that this escape response, like the feeding response to light, is likely caused by light’s generation of chemicals such as hydrogen peroxide.

The C. elegans worm has a very simple and thoroughly mapped nervous system consisting of 302 neurons, 20 of which are located in the pharynx, the feeding organ that ingests and grinds food. Bhatla found that one pair of pharyngeal neurons, known as the I2 neurons, controls the animal’s response to both light and hydrogen peroxide. A particular molecular receptor in that neuron, gustatory receptor 3 (GUR-3), and a molecularly similar receptor found in other neurons (LITE-1) are critical to the response. However, each receptor appears to function in a slightly different way.

GUR-3 detects hydrogen peroxide, whether it is found naturally in the environment or generated by light. There are many GUR-3 receptors in the I2 neuron, and through a mechanism that remains unknown, hydrogen peroxide stimulation of GUR-3 causes the pharynx to stop grinding. Another molecule called peroxiredoxin, an antioxidant, appears to help GUR-3 detect hydrogen peroxide.

While the GUR-3 receptor responds much more strongly to hydrogen peroxide than to light, the LITE-1 receptor is much more sensitive to light than to hydrogen peroxide. LITE-1 has previously been implicated in detecting light, but until now, it has been a mystery how a taste receptor could respond to light. The new study suggests that like GUR-3, LITE-1 indirectly senses light by detecting reactive oxygen compounds generated by light — including, but not limited to, hydrogen peroxide.

Kenneth Miller of the Oklahoma Medical Research Foundation published a paper in 2008 describing LITE-1 and hypothesizing that it might work by detecting a chemical product of light interaction. “This paper goes one step beyond that and identifies molecules that LITE-1 could be sensing to identify the presence of light,” says Miller, who was not part of the new study. “I thought it was a fascinating look at the complex gustatory sensory mechanism for molecules like hydrogen peroxide.”

Not found in humans

The molecular family of receptors that includes GUR-3 and LITE-1 is specific to invertebrates, and is not found in humans. However, peroxiredoxin is found in humans, particularly in the eye, so the researchers suspect that peroxiredoxin might play a role in detecting reactive oxygen species generated by light in the eye.

The researchers are now trying to figure out the exact mechanism of hydrogen peroxide detection: For example, how exactly do these gustatory receptors detect reactive oxygen compounds? The researchers are also working to identify the neural circuit diagram that defines how the I2 neurons interact with other neurons to control the worms’ feeding behavior. Such neural circuit diagrams should provide insight into how the brains of worms, and people, generate behavior.

The research was funded by the National Science Foundation, the National Institutes of Health, and the Howard Hughes Medical Institute.

Researchers find new actions of neurochemicals

Although the tiny roundworm Caenorhabditis elegans has only 302 neurons in its entire nervous system, studies of this simple animal have significantly advanced our understanding of human brain function because it shares many genes and neurochemical signaling molecules with humans. Now MIT researchers have found novel C. elegans neurochemical receptors, the discovery of which could lead to new therapeutic targets for psychiatric disorders if similar receptors are found in humans.

Dopamine and serotonin are members of a class of neurochemicals called biogenic amines, which function in neuronal circuitry throughout the brain. Many drugs used to treat psychiatric disorders, including depression and schizophrenia, target these signaling systems, as do cocaine and other drugs of abuse. Scientists have long known of a class of biogenic-amine receptors that are G protein-coupled receptors (GPCRs) and that, when activated, trigger a slow but long-lasting cascade of intracellular events that modulate nervous system activity.

A study in the July 3 issue of Science has found that in C. elegans these chemicals also act on receptors of a fundamentally different type. These receptors are chloride channels that open and close quickly in response to the binding of a neurochemical messenger. By allowing the passage of negatively charged chloride ions across the cell membrane, chloride channels can rapidly inactivate nerve cells.

“These results underscore the importance of determining whether, as in the C. elegans nervous system, a diversity of biogenic amine-gated chloride channels function in the human brain,” said H. Robert Horvitz of the McGovern Institute for Brain Research at MIT and senior author of the study. “If so, such channels might define novel therapeutic targets for neuropsychiatric disorders, such as depression and schizophrenia.”

In 2000, Horvitz’s group discovered that serotonin activates a chloride channel they called MOD-1, which inhibits neuronal activity in C. elegans.

In the current study, Niels Ringstad and Namiko Abe, a postdoctoral researcher and an undergraduate in Horvitz’s laboratory, respectively, looked for other ion channels that could be receptors for biogenic amines. Using both in vitro and in vivo methods, they surveyed the functions of 26 ion channels similar to MOD-1 and found three additional ion channels with an affinity for biogenic amines: dopamine activates one, serotonin another, and tyramine (the role of which in the human brain is unknown) a third. All three were chloride channels, like MOD-1.

“We now have four members of a family of chloride channels that can act as receptors for biogenic amines in the worm,” Ringstad said. “That these neurochemicals activate both GPCRs and ion channels means that they can have very complex actions in the nervous system, both as slow-acting neuromodulators and as fast-acting inhibitory neurotransmitters.”

It is unknown as yet whether an equivalent to this new class of worm receptor exists in the human brain, but Horvitz points out that worms have proved remarkably informative for providing insights into human biology. In 2002, Horvitz shared the Nobel Prize in Physiology or Medicine for the discovery based on studies of C. elegans of the mechanism of programmed cell death, a central feature of some neurodegenerative diseases and many other disorders in humans.

“Historically, studies of C. elegans have delineated mechanisms of neurotransmission that subsequently proved to be conserved in humans,” says Horvitz, the David H. Koch Professor of Biology at MIT and a Howard Hughes Medical Institute Investigator. “The next step is to look for chloride channels controlled by biogenic amines in mammalian neurons.”

This study was supported by the NIH, the Howard Hughes Medical Institute, the Life Sciences Research Foundation, and The Medical Foundation.